Ebook Principles of biochemistry (4th edition): Part 2

Continued part 1, part 2 of ebook Principles of biochemistry (4th edition) provide readers with content about: glycolysis, gluconeogenesis, and the pentose phosphate pathway; the metabolism of glycogen in animals; the citric acid cycle; fatty acid catabolism; amino acid oxidation and the production of urea; oxidative phosphorylation and photophosphorylation; carbohydrate biosynthesis in plants and bacteria; lipid biosynthesis; biosynthesis of amino acids, nucleotides, and related molecules; integration and hormonal regulation of mammalian metabolism;...

c h a p t e r

Glucose occupies a central position in the metabolism

of plants, animals, and many microorganisms It isrelatively rich in potential energy, and thus a good fuel;

the complete oxidation of glucose to carbon dioxide and

water proceeds with a standard free-energy change of

2,840 kJ/mol By storing glucose as a high molecular

weight polymer such as starch or glycogen, a cell can

stockpile large quantities of hexose units while

main-taining a relatively low cytosolic osmolarity When

en-ergy demands increase, glucose can be released from

these intracellular storage polymers and used to

pro-duce ATP either aerobically or anaerobically

Glucose is not only an excellent fuel, it is also a markably versatile precursor, capable of supplying ahuge array of metabolic intermediates for biosynthetic

re-reactions A bacterium such as Escherichia coli can

ob-tain from glucose the carbon skeletons for every aminoacid, nucleotide, coenzyme, fatty acid, or other meta-bolic intermediate it needs for growth A comprehen-sive study of the metabolic fates of glucose would en-compass hundreds or thousands of transformations Inanimals and vascular plants, glucose has three majorfates: it may be stored (as a polysaccharide or as su-crose); oxidized to a three-carbon compound (pyru-vate) via glycolysis to provide ATP and metabolic in-termediates; or oxidized via the pentose phosphate(phosphogluconate) pathway to yield ribose 5-phos-phate for nucleic acid synthesis and NADPH for reduc-tive biosynthetic processes (Fig 14–1)

Organisms that do not have access to glucose fromother sources must make it Photosynthetic organismsmake glucose by first reducing atmospheric CO2 totrioses, then converting the trioses to glucose Non-photosynthetic cells make glucose from simpler three-and four-carbon precursors by the process of gluconeo-genesis, effectively reversing glycolysis in a pathwaythat uses many of the glycolytic enzymes

In this chapter we describe the individual reactions

of glycolysis, gluconeogenesis, and the pentose phate pathway and the functional significance of eachpathway We also describe the various fates of thepyruvate produced by glycolysis; they include the fer-mentations that are used by many organisms in anaer-obic niches to produce ATP and that are exploited in-dustrially as sources of ethanol, lactic acid, and other

phos-GLYCOLYSIS, GLUCONEOGENESIS, AND THE PENTOSE PHOSPHATE

PATHWAY

14.1 Glycolysis 522

14.2 Feeder Pathways for Glycolysis 534

14.3 Fates of Pyruvate under Anaerobic Conditions:

Fermentation 538

14.4 Gluconeogenesis 543

14.5 Pentose Phosphate Pathway of Glucose

Oxidation 549

The problem of alcoholic fermentation, of the origin and

nature of that mysterious and apparently spontaneous

change, which converted the insipid juice of the grape

into stimulating wine, seems to have exerted a fascination

over the minds of natural philosophers from the very

O HO H

CH 2

OH H

commercially useful products And we look at the

path-ways that feed various sugars from mono-, di-, and

poly-saccharides into the glycolytic pathway The discussion

of glucose metabolism continues in Chapter 15, where

we describe the opposing anabolic and catabolic

path-ways that connect glucose and glycogen, and use the

processes of carbohydrate synthesis and degradation as

examples of the many mechanisms by which organisms

regulate metabolic pathways

14.1 Glycolysis

In glycolysis (from the Greek glykys, meaning “sweet,”

and lysis, meaning “splitting”), a molecule of glucose is

degraded in a series of enzyme-catalyzed reactions to

yield two molecules of the three-carbon compound

pyruvate During the sequential reactions of glycolysis,

some of the free energy released from glucose is

con-served in the form of ATP and NADH Glycolysis was

the first metabolic pathway to be elucidated and is

prob-ably the best understood From Eduard Buchner’s

dis-covery in 1897 of fermentation in broken extracts of

yeast cells until the elucidation of the whole pathway in

yeast (by Otto Warburg and Hans von Euler-Chelpin)

and in muscle (by Gustav Embden and Otto Meyerhof)

in the 1930s, the reactions of glycolysis in extracts ofyeast and muscle were a major focus of biochemical re-search The philosophical shift that accompanied thesediscoveries was announced by Jacques Loeb in 1906:Through the discovery of Buchner, Biology wasrelieved of another fragment of mysticism Thesplitting up of sugar into CO2and alcohol is nomore the effect of a “vital principle” than thesplitting up of cane sugar by invertase The history of this problem is instructive, as it warns

us against considering problems as beyond ourreach because they have not yet found their solution

The development of methods of enzyme tion, the discovery and recognition of the importance ofcoenzymes such as NAD, and the discovery of the piv-otal metabolic role of ATP and other phosphorylatedcompounds all came out of studies of glycolysis The gly-colytic enzymes of many species have long since beenpurified and thoroughly studied

purifica-Glycolysis is an almost universal central pathway ofglucose catabolism, the pathway with the largest flux ofcarbon in most cells The glycolytic breakdown of glu-cose is the sole source of metabolic energy in somemammalian tissues and cell types (erythrocytes, renalmedulla, brain, and sperm, for example) Some plant tis-sues that are modified to store starch (such as potatotubers) and some aquatic plants (watercress, for ex-ample) derive most of their energy from glycolysis;many anaerobic microorganisms are entirely dependent

on glycolysis

Fermentation is a general term for the anaerobic

degradation of glucose or other organic nutrients to tain energy, conserved as ATP Because living organismsfirst arose in an atmosphere without oxygen, anaerobicbreakdown of glucose is probably the most ancient bio-logical mechanism for obtaining energy from organicfuel molecules In the course of evolution, the chemistry

ob-of this reaction sequence has been completely served; the glycolytic enzymes of vertebrates are closely

con-similar, in amino acid sequence andthree-dimensional structure, to theirhomologs in yeast and spinach Gly-colysis differs among species only inthe details of its regulation and in thesubsequent metabolic fate of thepyruvate formed The thermodynamicprinciples and the types of regulatorymechanisms that govern glycolysis arecommon to all pathways of cell me-tabolism A study of glycolysis cantherefore serve as a model for manyaspects of the pathways discussedthroughout this book

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

522

Glycogen, starch, sucrose

oxidation via pentose phosphate pathway

oxidation via glycolysis

Glucose

storage

FIGURE 14–1 Major pathways of glucose utilization Although not

the only possible fates for glucose, these three pathways are the most

significant in terms of the amount of glucose that flows through them

in most cells.

Hans von Euler-Chelpin,

1873–1964

Gustav Embden, 1874–1933

Otto Meyerhof, 1884–1951

Before examining each step of the pathway in somedetail, we take a look at glycolysis as a whole.

An Overview: Glycolysis Has Two Phases

The breakdown of the six-carbon glucose into two

mol-ecules of the three-carbon pyruvate occurs in ten steps,

the first five of which constitute the preparatory phase

(Fig 14–2a) In these reactions, glucose is first

phos-phorylated at the hydroxyl group on C-6 (step 1 ) The

D-glucose 6-phosphate thus formed is converted to D

-fructose 6-phosphate (step 2 ), which is again

phos-phorylated, this time at C-1, to yield D-fructose

1,6-bisphosphate (step 3 ) For both phosphorylations, ATP

is the phosphoryl group donor As all sugar derivatives

in glycolysis are the Disomers, we will usually omit the

Ddesignation except when emphasizing stereochemistry

Fructose 1,6-bisphosphate is split to yield twothree-carbon molecules, dihydroxyacetone phosphate

and glyceraldehyde 3-phosphate (step 4 ); this is the

“lysis” step that gives the pathway its name The

dihy-droxyacetone phosphate is isomerized to a second

mol-ecule of glyceraldehyde 3-phosphate (step 5 ), ending

the first phase of glycolysis From a chemical

perspec-tive, the isomerization in step 2 is critical for setting

up the phosphorylation and COC bond cleavage

reac-tions in steps 3 and 4 , as detailed later Note that two

molecules of ATP are invested before the cleavage of

glucose into two three-carbon pieces; later there will be

a good return on this investment To summarize: in the

preparatory phase of glycolysis the energy of ATP is

invested, raising the free-energy content of the

inter-mediates, and the carbon chains of all the metabolized

hexoses are converted into a common product,

glyceraldehyde 3-phosphate

The energy gain comes in the payoff phase of

gly-colysis (Fig 14–2b) Each molecule of glyceraldehyde

3-phosphate is oxidized and phosphorylated by

inor-ganic phosphate (not by ATP) to form

1,3-bisphospho-glycerate (step 6 ) Energy is then released as the two

molecules of 1,3-bisphosphoglycerate are converted to

two molecules of pyruvate (steps 7 through 10) Much

of this energy is conserved by the coupled

phosphory-lation of four molecules of ADP to ATP The net yield is

two molecules of ATP per molecule of glucose used,

be-cause two molecules of ATP were invested in the

preparatory phase Energy is also conserved in the

pay-off phase in the formation of two molecules of NADH

per molecule of glucose

In the sequential reactions of glycolysis, three types

of chemical transformations are particularly noteworthy:

(1) degradation of the carbon skeleton of glucose to

yield pyruvate, (2) phosphorylation of ADP to ATP

by high-energy phosphate compounds formed during

glycolysis, and (3) transfer of a hydride ion to NAD
,

to yield the acetyl group of acetyl-coenzyme A; theacetyl group is then oxidized completely to CO2by thecitric acid cycle (Chapter 16) The electrons from theseoxidations are passed to O2through a chain of carriers

in the mitochondrion, to form H2O The energy from theelectron-transfer reactions drives the synthesis of ATP

in the mitochondrion (Chapter 19)

The second route for pyruvate is its reduction to

lactate via lactic acid fermentation When vigorously

contracting skeletal muscle must function under

low-oxygen conditions (hypoxia), NADH cannot be

reoxi-dized to NAD
, but NAD
is required as an electron ceptor for the further oxidation of pyruvate Under theseconditions pyruvate is reduced to lactate, acceptingelectrons from NADH and thereby regenerating theNAD
necessary for glycolysis to continue Certain tis-sues and cell types (retina and erythrocytes, for exam-ple) convert glucose to lactate even under aerobic con-ditions, and lactate is also the product of glycolysisunder anaerobic conditions in some microorganisms(Fig 14–3)

ac-The third major route of pyruvate catabolism leads

to ethanol In some plant tissues and in certain tebrates, protists, and microorganisms such as brewer’syeast, pyruvate is converted under hypoxic or anaero-bic conditions into ethanol and CO2, a process called

inver-ethanol (alcohol) fermentation (Fig 14–3)

The oxidation of pyruvate is an important catabolicprocess, but pyruvate has anabolic fates as well It can,for example, provide the carbon skeleton for the syn-thesis of the amino acid alanine We return to these an-abolic reactions of pyruvate in later chapters

ATP Formation Coupled to Glycolysis During glycolysissome of the energy of the glucose molecule is conserved

in ATP, while much remains in the product, pyruvate.The overall equation for glycolysis is

Glucose
2NAD

2ADP
2P i88n

2 pyruvate
2NADH
2H

2ATP
2H 2 O (14–1)

For each molecule of glucose degraded to pyruvate, twomolecules of ATP are generated from ADP and Pi Wecan now resolve the equation of glycolysis into twoprocesses—the conversion of glucose to pyruvate,which is exergonic:

Glucose
2NAD
88n2 pyruvate
2NADH
2H
(14–2)

G  146 kJ/mol

14.1 Glycolysis 523

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

524

O cleavage

of 6-carbon sugar phosphate to two 3-carbon sugar phosphates

5

first priming reaction

2 1

HO H

OH O

H

CH 2

HO H

C

O

O

CH2O

Payoff phase

Oxidative conversion of glyceraldehyde 3-phosphate to pyruvate and the coupled formation of ATP and NADH

C CH2OH O

Glyceraldehyde 3-phosphate

ADP

second priming reaction

CH2CH O

2H 2 O 2-Phosphoglycerate (2)

Phosphoenolpyruvate (2)

first forming reaction (substrate-level phosphorylation)

ATP-6

7

9 8

4

O

C O

Preparatory phase

Phosphorylation of glucose and its conversion to glyceraldehyde 3-phosphate

(a)

(b)



oxidation and phosphorylation

C O

H

CH2 CH O

P

OH

C

CH 2 CH O

P

OH

CH2 CH OH

CH 2

C

1 2 3 4 5 6

NADH 2 ATP

2 ATP

OH

OH

C O

H

O

O C

O

O

O
O

O
O

1 2

3

4 5

Hexokinase

Phosphohexose isomerase

fructokinase-1

Phospho-Aldolase

Triose phosphate isomerase

6

7

8

9 10

Glyceraldehyde 3-phosphate dehydrogenase Phospho- glycerate kinase Phospho- glycerate mutase Enolase Pyruvate kinase

FIGURE 14–2 The two phases of glycolysis For each molecule of

glu-cose that passes through the preparatory phase (a), two molecules of

glyceraldehyde 3-phosphate are formed; both pass through the payoff

phase (b) Pyruvate is the end product of the second phase of

glycol-ysis For each glucose molecule, two ATP are consumed in the

prepara-tory phase and four ATP are produced in the payoff phase, giving a

net yield of two ATP per molecule of glucose converted to pyruvate The numbered reaction steps are catalyzed by the enzymes listed on the right, and also correspond to the numbered headings in the text discussion Keep in mind that each phosphoryl group, represented here as P , has two negative charges (OPO 3
).

and the formation of ATP from ADP and Pi, which is

endergonic:

2ADP  2P i88n2ATP  2H 2 O (14–3)

G2   2(30.5 kJ/mol)  61.0 kJ/mol

The sum of Equations 14–2 and 14–3 gives the overall

standard free-energy change of glycolysis, Gs:

Gs  G1  G2  
146 kJ/mol  61.0 kJ/mol


85 kJ/mol

Under standard conditions and in the cell, glycolysis is

an essentially irreversible process, driven to completion

by a large net decrease in free energy At the actual

in-tracellular concentrations of ATP, ADP, and Pi(see Box

13–1) and of glucose and pyruvate, the energy released

in glycolysis (with pyruvate as the end product) is

re-covered as ATP with an efficiency of more than 60%

Energy Remaining in Pyruvate Glycolysis releases only a

small fraction of the total available energy of the

glu-cose molecule; the two molecules of pyruvate formed

by glycolysis still contain most of the chemical

poten-tial energy of glucose, energy that can be extracted by

oxidative reactions in the citric acid cycle (Chapter 16)

and oxidative phosphorylation (Chapter 19)

Importance of Phosphorylated Intermediates Each of the

nine glycolytic intermediates between glucose and

pyru-vate is phosphorylated (Fig 14–2) The phosphoryl

groups appear to have three functions

1. Because the plasma membrane generally lacks

transporters for phosphorylated sugars, the phorylated glycolytic intermediates cannot leavethe cell After the initial phosphorylation, no fur-ther energy is necessary to retain phosphorylatedintermediates in the cell, despite the large differ-ence in their intracellular and extracellular con-centrations

phos-2. Phosphoryl groups are essential components in

the enzymatic conservation of metabolic energy

Energy released in the breakage of dride bonds (such as those in ATP) is partiallyconserved in the formation of phosphate esterssuch as glucose 6-phosphate High-energy phos-phate compounds formed in glycolysis (1,3-bisphos-phoglycerate and phosphoenolpyruvate) donatephosphoryl groups to ADP to form ATP

phosphoanhy-3. Binding energy resulting from the binding of

phos-phate groups to the active sites of enzymes lowersthe activation energy and increases the specificity

of the enzymatic reactions (Chapter 6) The phate groups of ADP, ATP, and the glycolytic in-termediates form complexes with Mg2, and thesubstrate binding sites of many glycolytic enzymesare specific for these Mg2complexes Most gly-colytic enzymes require Mg2for activity

phos-The Preparatory Phase of Glycolysis Requires ATP

In the preparatory phase of glycolysis, two molecules ofATP are invested and the hexose chain is cleaved into

two triose phosphates The realization that lated hexoses were intermediates in glycolysis came

phosphory-slowly and serendipitously In 1906, Arthur Harden andWilliam Young tested their hypothesis that inhibitors ofproteolytic enzymes would stabilize the glucose-fermenting enzymes in yeast extract They added bloodserum (known to contain inhibitors of proteolytic en-zymes) to yeast extracts and observed the predictedstimulation of glucose metabolism However, in a con-trol experiment intended to show that boiling the serumdestroyed the stimulatory activity, they discovered thatboiled serum was just as effective at stimulating glycol-ysis Careful examination and testing of the contents of

aerobic conditions 2CO2

citric acid cycle

Fermentation to lactate in vigor- ously contracting muscle, in erythro- cytes, in some other cells, and

in some organisms

micro-anaerobic conditions

hypoxic or anaerobic conditions

Animal, plant, and many microbial cells under aerobic conditions

Fermentation to ethanol

in yeast

FIGURE 14–3 Three possible catabolic fates of the pyruvate formed

in glycolysis Pyruvate also serves as a precursor in many anabolic

re-actions, not shown here.

Arthur Harden, 1865–1940

William Young, 1878–1942

the boiled serum revealed that inorganic phosphate was

responsible for the stimulation Harden and Young soon

discovered that glucose added to their yeast extract was

converted to a hexose bisphosphate (the

“Harden-Young ester,” eventually identified as fructose

1,6-bisphosphate) This was the beginning of a long series

of investigations on the role of organic esters of

phos-phate in biochemistry, which has led to our current

un-derstanding of the central role of phosphoryl group

transfer in biology

1 Phosphorylation of Glucose In the first step of

glycol-ysis, glucose is activated for subsequent reactions by its

phosphorylation at C-6 to yield glucose 6-phosphate,

with ATP as the phosphoryl donor:

This reaction, which is irreversible under

intracel-lular conditions, is catalyzed by hexokinase Recall that

kinases are enzymes that catalyze the transfer of the

terminal phosphoryl group from ATP to an acceptor

nu-cleophile (see Fig 13–10) Kinases are a subclass of

transferases (see Table 6–3) The acceptor in the case

of hexokinase is a hexose, normally D-glucose, although

hexokinase also catalyzes the phosphorylation of other

common hexoses, such as D-fructose and D-mannose

Hexokinase, like many other kinases, requires Mg2

for its activity, because the true substrate of the enzyme

is not ATP4
but the MgATP2
complex (see Fig 13–2)

Mg2 shields the negative charges of the phosphoryl

groups in ATP, making the terminal phosphorus atom an

easier target for nucleophilic attack by an OOH of

glu-cose Hexokinase undergoes a profound change in

shape, an induced fit, when it binds glucose; two

do-mains of the protein move about 8 Å closer to each other

when ATP binds (see Fig 6–22) This movement brings

bound ATP closer to a molecule of glucose also bound

to the enzyme and blocks the access of water (from the

solvent), which might otherwise enter the active site

and attack (hydrolyze) the phosphoanhydride bonds of

ATP Like the other nine enzymes of glycolysis,

hexo-kinase is a soluble, cytosolic protein

Hexokinase is present in all cells of all organisms

Hepatocytes also contain a form of hexokinase called

hexokinase IV or glucokinase, which differs from other

forms of hexokinase in kinetic and regulatory

proper-ties (see Box 15–2) Two enzymes that catalyze the

O OPO3

H OH

H H

OH H

CH2

OH

H OH

H

H

OH H

2 3

same reaction but are encoded in different genes are

called isozymes.

2 Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate

The enzyme phosphohexose isomerase

(phospho-glucose isomerase) catalyzes the reversible

isomer-ization of glucose 6-phosphate, an aldose, to fructose

6-phosphate, a ketose:

The mechanism for this reaction is shown in Figure14–4 The reaction proceeds readily in either direction,

as might be expected from the relatively small change

in standard free energy This isomerization has a cal role in the overall chemistry of the glycolytic path-way, as the rearrangement of the carbonyl and hydroxylgroups at C-1 and C-2 is a necessary prelude to the nexttwo steps The phosphorylation that occurs in the nextreaction (step 3 ) requires that the group at C-1 first

criti-be converted from a carbonyl to an alcohol, and in thesubsequent reaction (step 4 ) cleavage of the bond be-tween C-3 and C-4 requires a carbonyl group at C-2 (p 485)

3 Phosphorylation of Fructose 6-Phosphate to Fructose Bisphosphate In the second of the two priming reactions

1,6-of glycolysis, phosph1,6-ofructokinase-1 (PFK-1)

cat-alyzes the transfer of a phosphoryl group from ATP to

fructose 6-phosphate to yield fructose

1,6-bisphos-phate:

OPO3

phosphofructokinase-1 (PFK-1)

Mg2

O

HO

H H

CH2

OH OH

OH H

H H

OH H

4

2 1

6 5

CH 2 OPO 3

CH2OPO3

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

526

This enzyme is called PFK-1 to distinguish it from a

sec-ond enzyme (PFK-2) that catalyzes the formation of

fructose 2,6-bisphosphate from fructose 6-phosphate in

a separate pathway The PFK-1 reaction is essentially

irreversible under cellular conditions, and it is the first

“committed” step in the glycolytic pathway; glucose

6-phosphate and fructose 6-phosphate have other

pos-sible fates, but fructose 1,6-bisphosphate is targeted for

glycolysis

Some bacteria and protists and perhaps all plantshave a phosphofructokinase that uses pyrophosphate

(PPi), not ATP, as the phosphoryl group donor in the

synthesis of fructose 1,6-bisphosphate:

major point of regulation in glycolysis The activity of

PFK-1 is increased whenever the cell’s ATP supply is

depleted or when the ATP breakdown products, ADP

and AMP (particularly the latter), are in excess The

en-zyme is inhibited whenever the cell has ample ATP and

is well supplied by other fuels such as fatty acids In

some organisms, fructose 2,6-bisphosphate (not to be

confused with the PFK-1 reaction product, fructose

1,6-bisphosphate) is a potent allosteric activator of PFK-1

The regulation of this step in glycolysis is discussed in

greater detail in Chapter 15

4 Cleavage of Fructose 1,6-Bisphosphate The enzyme

fructose 1,6-bisphosphate aldolase, often called

simply aldolase, catalyzes a reversible aldol

condensa-tion (p 485) Fructose 1,6-bisphosphate is cleaved to

yield two different triose phosphates, glyceraldehyde

phosphate, a ketose:

There are two classes of aldolases Class I aldolases,found in animals and plants, use the mechanism shown

in Figure 14–5 Class II enzymes, in fungi and bacteria,

do not form the Schiff base intermediate Instead, a zincion at the active site is coordinated with the carbonyloxygen at C-2; the Zn2
polarizes the carbonyl group

CHOH

Glyceraldehyde 3-phosphate

O H H

OH

Fructose 1,6-bisphosphate HO

1

(1) 2

(2) 5

(5)

4

(4) 3

(3) 6

Phosphohexose

isomerase

binding and ring opening

O

HO H H

1 CH2OH

OH Fructose 6-phosphate

:

O H B

HO 3 CH

2 C

1 C

OH H+H

HCOH HCOH

cis-Enediol

intermediate

OH H BH

HOCH C

C

HCOH HCOH

MECHANISM FIGURE 14–4 The phosphohexose isomerase reaction The ring

opening and closing reactions (steps 1 and

4 ) are catalyzed by an active-site His residue, by mechanisms omitted here for simplicity The movement of the proton between C-2 and C-1 (steps 2 and 3 ) is base-catalyzed by an active-site Glu residue (shown as B:) The proton (pink) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and the nearby hydroxyl group After its transfer from C-2 to the active-site Glu residue, the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step 2 is not necessarily the same one that is added to C-1

in step 3 (The additional exchange of protons (yellow and blue) between the hydroxyl groups and solvent is shown for completeness The hydroxyl groups are weak acids and can exchange protons with the surrounding water whether the isomerization reaction is underway or not.)

Phosphohexose Isomerase Mechanism

and stabilizes the enolate intermediate created in the

COC bond cleavage step

Although the aldolase reaction has a strongly

posi-tive standard free-energy change in the direction of

fruc-tose 1,6-bisphosphate cleavage, at the lower

concentra-tions of reactants present in cells, the actual free-energy

change is small and the aldolase reaction is readily

versible We shall see later that aldolase acts in the

re-verse direction during the process of gluconeogenesis(see Fig 14–16)

5 Interconversion of the Triose Phosphates Only one of thetwo triose phosphates formed by aldolase, glyceralde-hyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis The other product, dihy-droxyacetone phosphate, is rapidly and reversibly

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

528

Aldolase

binding and ring opening

:

:

H N H Lys HOCH

B BH

Enamine intermediate

Proton exchange with solution restores enzyme

first product released

second product released

N +

H

C H

H HO

O H C HCOH

Glyceraldehyde 3-phosphate

CH2OPO3

2–

acetone phosphate

Dihydroxy-CH2OH

4

Protonated Schiff base

Protonated Schiff base

HO H H

OH O

MECHANISM FIGURE 14–5 The class I aldolase reaction The

reac-tion shown here is the reverse of an aldol condensareac-tion Note that

cleavage between C-3 and C-4 depends on the presence of the

car-bonyl group at C-2 1 and 2 The carcar-bonyl reacts with an active-site

Lys residue to form an imine, which stabilizes the carbanion generated

by the bond cleavage—an imine delocalizes electrons even better than

does a carbonyl 3 Bond cleavage releases glyceraldeyde 3-phosphate

as the first product 4 The resulting enamine covalently linked to the enzyme is isomerized to a protonated Schiff base, and 5 hydrolysis

of the Schiff base generates dihydroxyacetone phosphate as the ond product A and B represent amino acid residues that serve as general acid (A) or base (B).

sec-converted to glyceraldehyde 3-phosphate by the fifth

enzyme of the sequence, triose phosphate isomerase:

The reaction mechanism is similar to the reaction

pro-moted by phosphohexose isomerase in step 2 of

gly-colysis (Fig 14–4) After the triose phosphate isomerase

reaction, C-1, C-2, and C-3 of the starting glucose are

chemically indistinguishable from C-6, C-5, and C-4,

re-spectively (Fig 14–6), setting up the efficient

metabo-lism of the entire six-carbon glucose molecule

This reaction completes the preparatory phase ofglycolysis The hexose molecule has been phosphory-

lated at C-1 and C-6 and then cleaved to form two

mol-ecules of glyceraldehyde 3-phosphate

The Payoff Phase of Glycolysis Yields ATP and NADH

The payoff phase of glycolysis (Fig 14–2b) includes the

energy-conserving phosphorylation steps in which some

of the free energy of the glucose molecule is conserved

in the form of ATP Remember that one molecule of

glu-cose yields two molecules of glyceraldehyde

3-phos-phate; both halves of the glucose molecule follow the

same pathway in the second phase of glycolysis Theconversion of two molecules of glyceraldehyde 3-phos-phate to two molecules of pyruvate is accompanied bythe formation of four molecules of ATP from ADP How-ever, the net yield of ATP per molecule of glucose de-graded is only two, because two ATP were invested inthe preparatory phase of glycolysis to phosphorylate thetwo ends of the hexose molecule

6 Oxidation of Glyceraldehyde 3-Phosphate to phoglycerate The first step in the payoff phase is the

1,3-Bisphos-oxidation of glyceraldehyde 3-phosphate to

1,bis-phosphoglycerate, catalyzed by glyceraldehyde phosphate dehydrogenase:

OH

OH H

6

1 2

3 4

4 or 3

5 or 2

6 or 1

4 5 6

Derived from glucose carbons Fructose 1,6-bisphosphate

triose phosphate isomerase

H HO

CH2C C O

O

1 2

3

H

P

1 2 3

Glyceraldehyde 3-phosphate

(a)

P

D -Glyceraldehyde 3-phosphate

re-3-phosphate (two molecules) (b) Each carbon of glyceraldehyde

3-phosphate is derived from either of two specific carbons of glucose Note that the numbering of the carbon atoms of glyceraldehyde 3-phosphate differs from that of the glucose from which it is derived.

In glyceraldehyde 3-phosphate, the most complex functional group (the carbonyl) is specified as C-1 This numbering change is important for in- terpreting experiments with glucose in which a single carbon is labeled with a radioisotope (See Problems 3 and 5 at the end of this chapter.)

C

OPO 3

Inorganic phosphate O

2

O P HO





Glyceraldehyde 3-phosphate

7.5 kJ/mol

DG

HCOH

Glyceraldehyde 3-phosphate

triose phosphate isomerase

C

CH2O

H OH

C

O

Dihydroxyacetone phosphate

This is the first of the two energy-conserving reactions

of glycolysis that eventually lead to the formation of ATP

The aldehyde group of glyceraldehyde 3-phosphate is

oxidized, not to a free carboxyl group but to a carboxylic

acid anhydride with phosphoric acid This type of

an-hydride, called an acyl phosphate, has a very high

stan-dard free energy of hydrolysis (
G  49.3 kJ/mol;

see Fig 13–4, Table 13–6) Much of the free energy of

oxidation of the aldehyde group of glyceraldehyde

3-phosphate is conserved by formation of the acyl

phos-phate group at C-1 of 1,3-bisphosphoglycerate

The acceptor of hydrogen in the glyceraldehyde

3-phosphate dehydrogenase reaction is NAD (see Fig

13–15), bound to a Rossmann fold as shown in Figure

13–16 The reduction of NAD proceeds by the

enzy-matic transfer of a hydride ion (:H) from the aldehyde

group of glyceraldehyde 3-phosphate to the

nicoti-namide ring of NAD, yielding the reduced coenzymeNADH The other hydrogen atom of the substrate mol-ecule is released to the solution as H

Glyceraldehyde 3-phosphate is covalently bound tothe dehydrogenase during the reaction (Fig 14–7) Thealdehyde group of glyceraldehyde 3-phosphate reactswith the OSH group of an essential Cys residue in theactive site, in a reaction analogous to the formation of a

hemiacetal (see Fig 7–5), in this case producing a

thio-hemiacetal Reaction of the essential Cys residue with aheavy metal such as Hg2 irreversibly inhibits the enzyme.Because cells maintain only limited amounts ofNAD, glycolysis would soon come to a halt if the NADHformed in this step of glycolysis were not continuouslyreoxidized The reactions in which NADis regeneratedanaerobically are described in detail in Section 14.3, inour discussion of the alternative fates of pyruvate

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

530

C

HCOH O

: N NH

His

formation of thiohemiacetal intermediate

C HCOH

CH2OPO32–

NAD+

S Cys

: N NH

His

3 oxidation tothioester intermediate

NADH

S Cys

C

HCOH O

CH2OPO32–

NAD +

S Cys

C

HCOH O

CH2OPO32–

CH2OPO32–

OPO32–

O – – O P OH O

Glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate

formation of substrate complex

enzyme-4

NADH exchanged for NAD + ; attack

NH

His +

NH

His +

NH

His +

MECHANISM FIGURE 14–7 The glyceraldehyde 3-phosphate

dehy-drogenase reaction After 1 formation of the enzyme-substrate

com-plex, 2 a covalent thiohemiacetal linkage forms between the

sub-strate and the OSH group of a Cys residue—facilitated by acid-base

catalysis with a neighboring base catalyst, probably a His residue

3 This enzyme-substrate intermediate is oxidized by NADbound

to the active site, forming a covalent acyl-enzyme intermediate, a

thioester 4 The newly formed NADH leaves the active site and is replaced by another NAD molecule The bond between the acyl group and the thiol group of the enzyme has a very high standard free energy of hydrolysis 5 This bond undergoes phosphorolysis (attack

by Pi), releasing the acyl phosphate product, 1,3-bisphosphoglycerate Formation of this product conserves much of the free energy liberated during oxidation of the aldehyde group of glyceraldehyde 3-phosphate.

7 Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP

The enzyme phosphoglycerate kinase transfers the

high-energy phosphoryl group from the carboxyl group

of 1,bisphosphoglycerate to ADP, forming ATP and

3-phosphoglycerate:

Notice that [H
] is not included in Q In biochemical

cal-culations, [H
] is assumed to be a constant (107M),and this constant is included in the definition of G

(p 491)

When the mass-action ratio is less than 1.0, its ural logarithm has a negative sign Step 7 , by consum-ing the product of step 6 (1,3-bisphosphoglycerate),keeps [1,3-bisphosphoglycerate] relatively low in the

nat-steady state and thereby keeps Q for the overall coupling process small When Q is small, the contribution

energy-of ln Q can make G strongly negative This is simply

another way of showing how the two reactions, steps

6 and 7 , are coupled through a common intermediate.The outcome of these coupled reactions, both re-versible under cellular conditions, is that the energy re-leased on oxidation of an aldehyde to a carboxylategroup is conserved by the coupled formation of ATPfrom ADP and Pi The formation of ATP by phosphorylgroup transfer from a substrate such as 1,3-bisphos-

phoglycerate is referred to as a substrate-level

phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation Substrate-level

phosphorylations involve soluble enzymes and chemicalintermediates (1,3-bisphosphoglycerate in this case).Respiration-linked phosphorylations, on the other hand,involve membrane-bound enzymes and transmembranegradients of protons (Chapter 19)

8 Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

The enzyme phosphoglycerate mutase catalyzes a

re-versible shift of the phosphoryl group between C-2 andC-3 of glycerate; Mg2
is essential for this reaction:

The reaction occurs in two steps (Fig 14–8) A phoryl group initially attached to a His residue of themutase is transferred to the hydroxyl group at C-2 of 3-phosphoglycerate, forming 2,3-bisphosphoglycerate(2,3-BPG) The phosphoryl group at C-3 of 2,3-BPG isthen transferred to the same His residue, producing 2-phosphoglycerate and regenerating the phosphorylatedenzyme Phosphoglycerate mutase is initially phospho-rylated by phosphoryl transfer from 2,3-BPG, which isrequired in small quantities to initiate the catalytic cy-cle and is continuously regenerated by that cycle Al-though in most cells 2,3-BPG is present in only traceamounts, it is a major component (~5 mM) of erythro-cytes, where it regulates the affinity of hemoglobin for

phos-14.1 Glycolysis 531

O

P

O O

C HCOH

Mg 2 phosphoglycerate kinase

OPO32

C HCOH

P

G  18.5 kJ/mol

Notice that phosphoglycerate kinase is named for the

reverse reaction Like all enzymes, it catalyzes the

re-action in both directions This enzyme acts in the

di-rection suggested by its name during gluconeogenesis

(see Fig 14–16) and during photosynthetic CO2

assim-ilation (see Fig 20–4)

Steps 6 and 7 of glycolysis together constitute anenergy-coupling process in which 1,3-bisphosphoglyc-

erate is the common intermediate; it is formed in the

first reaction (which would be endergonic in isolation),

and its acyl phosphate group is transferred to ADP in

the second reaction (which is strongly exergonic) The

sum of these two reactions is

Glyceraldehyde 3-phosphate
ADP
P i
NAD

3-phosphoglycerate
ATP
NADH
H

G  12.5 kJ/mol

Thus the overall reaction is exergonic

Recall from Chapter 13 that the actual free-energychange, G, is determined by the standard free-energy

change, G, and the mass-action ratio, Q, which is the

ratio [products]/[reactants] (see Eqn 13–3) For step 6

CH 2

O

Mg 2
phosphoglycerate mutase

O

O

C HC

PO 3 

O

oxygen (see Fig 5–17; note that in the context of

he-moglobin regulation, 2,3-bisphosphoglycerate is usually

abbreviated as simply BPG)

9 Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

In the second glycolytic reaction that generates a

com-pound with high phosphoryl group transfer potential,

enolase promotes reversible removal of a molecule of

water from 2-phosphoglycerate to yield

phospho-enolpyruvate (PEP):

The mechanism of the enolase reaction is presented in

Figure 6–23 Despite the relatively small standard

free-energy change of this reaction, there is a very large

difference in the standard free energy of hydrolysis of

the phosphoryl groups of the reactant and product:

17.6 kJ/mol for 2-phosphoglycerate (a low-energy phate ester) and
61.9 kJ/mol for phosphoenolpyruvate(a compound with a very high standard free energy

phos-of hydrolysis) (see Fig 13–3, Table 13–6) Although2-phosphoglycerate and phosphoenolpyruvate contain

nearly the same total amount of energy, the loss of the

water molecule from 2-phosphoglycerate causes a distribution of energy within the molecule, greatlyincreasing the standard free energy of hydrolysis of thephosphoryl group

re-10 Transfer of the Phosphoryl Group from vate to ADP The last step in glycolysis is the transfer ofthe phosphoryl group from phosphoenolpyruvate to

Phosphoenolpyru-ADP, catalyzed by pyruvate kinase, which requires K

and either Mg2or Mn2:

In this substrate-level phosphorylation, the product

pyruvate first appears in its enol form, then

tautomer-izes rapidly and nonenzymatically to its keto form, whichpredominates at pH 7:

The overall reaction has a large, negative standard energy change, due in large part to the spontaneous con-version of the enol form of pyruvate to the keto form(see Fig 13–3) The G of phosphoenolpyruvate

Pyruvate (keto form)

CH 2 OPO 3

3-Phosphoglycerate

COO
HCOPO 3

CH 2 OPO 3

2,3-Bisphosphoglycerate

(2,3-BPG)

COO
HCOPO 3

CH 2 OH 2-Phosphoglycerate

His



Phosphoglycerate mutase 1

2

His

FIGURE 14–8 The phosphoglycerate mutase reaction The enzyme is

initially phosphorylated on a His residue 1 The phosphoenzyme

transfers its phosphoryl group to 3-phosphoglycerate, forming

2,3-BPG 2 The phosphoryl group at C-3 of 2,3-BPG is transferred to the

same His residue on the enzyme, producing 2-phosphoglycerate and

regenerating the phosphoenzyme.

P

O

O P

, K pyruvate kinase

O

O

C C

CH 2

P P

Adenine Rib

hydrolysis is
61.9 kJ/mol; about half of this energy is

conserved in the formation of the phosphoanhydride

bond of ATP (G 
30.5 kJ/mol), and the rest

(
31.4 kJ/mol) constitutes a large driving force

push-ing the reaction toward ATP synthesis The pyruvate

kinase reaction is essentially irreversible under

intra-cellular conditions and is an important site of

regula-tion, as described in Chapter 15

The Overall Balance Sheet Shows a Net Gain of ATP

We can now construct a balance sheet for glycolysis to

account for (1) the fate of the carbon skeleton of

glu-cose, (2) the input of Piand ADP and the output of ATP,

and (3) the pathway of electrons in the

oxidation-reduction reactions The left-hand side of the following

equation shows all the inputs of ATP, NAD, ADP, and

Pi(consult Fig 14–2), and the right-hand side shows all

the outputs (keep in mind that each molecule of glucose

yields two molecules of pyruvate):

Glucose  2ATP  2NAD   4ADP  2P i88n

2 pyruvate  2ADP  2NADH  2H   4ATP  2H 2 O

Canceling out common terms on both sides of the

equa-tion gives the overall equaequa-tion for glycolysis under

aer-obic conditions:

Glucose  2NAD   2ADP  2P i88n

2 pyruvate  2NADH  2H   2ATP  2H 2 O

The two molecules of NADH formed by glycolysis

in the cytosol are, under aerobic conditions, reoxidized

to NADby transfer of their electrons to the

electron-transfer chain, which in eukaryotic cells is located in the

mitochondria The electron-transfer chain passes these

electrons to their ultimate destination, O2:

2NADH  2H   O 288n2NAD 2H 2 O

Electron transfer from NADH to O2in mitochondria

pro-vides the energy for synthesis of ATP by

respiration-linked phosphorylation (Chapter 19)

In the overall glycolytic process, one molecule ofglucose is converted to two molecules of pyruvate (the

pathway of carbon) Two molecules of ADP and two of

Piare converted to two molecules of ATP (the pathway

of phosphoryl groups) Four electrons, as two hydride

ions, are transferred from two molecules of

glyceralde-hyde 3-phosphate to two of NAD(the pathway of

elec-trons)

Glycolysis Is under Tight Regulation

During his studies on the fermentation of glucose by

yeast, Louis Pasteur discovered that both the rate and

the total amount of glucose consumption were many

times greater under anaerobic than aerobic conditions

Later studies of muscle showed the same large

differ-ence in the rates of anaerobic and aerobic glycolysis.The biochemical basis of this “Pasteur effect” is nowclear The ATP yield from glycolysis under anaerobicconditions (2 ATP per molecule of glucose) is muchsmaller than that from the complete oxidation of glu-cose to CO2under aerobic conditions (30 or 32 ATP perglucose; see Table 19–5) About 15 times as much glu-cose must therefore be consumed anaerobically as aer-obically to yield the same amount of ATP

The flux of glucose through the glycolytic pathway

is regulated to maintain nearly constant ATP levels (aswell as adequate supplies of glycolytic intermediatesthat serve biosynthetic roles) The required adjustment

in the rate of glycolysis is achieved by a complex play among ATP consumption, NADH regeneration, andallosteric regulation of several glycolytic enzymes—in-cluding hexokinase, PFK-1, and pyruvate kinase—and

inter-by second-to-second fluctuations in the concentration

of key metabolites that reflect the cellular balance tween ATP production and consumption On a slightlylonger time scale, glycolysis is regulated by the hor-mones glucagon, epinephrine, and insulin, and bychanges in the expression of the genes for several gly-colytic enzymes We return to a more detailed discus-sion of the regulation of glycolysis in Chapter 15

be-Cancerous Tissue Has Deranged Glucose Catabolism

Glucose uptake and glycolysis proceed about tentimes faster in most solid tumors than in non-cancerous tissues Tumor cells commonly experiencehypoxia (limited oxygen supply), because they initiallylack an extensive capillary network to supply the tumorwith oxygen As a result, cancer cells more than 100 to

200
m from the nearest capillaries depend on

anaero-bic glycolysis for much of their ATP production Theytake up more glucose than normal cells, converting it topyruvate and then to lactate as they recycle NADH Thehigh glycolytic rate may also result in part from smallernumbers of mitochondria in tumor cells; less ATP made

by respiration-linked phosphorylation in mitochondriameans more ATP is needed from glycolysis In addition,some tumor cells overproduce several glycolytic en-zymes, including an isozyme of hexokinase that associ-ates with the cytosolic face of the mitochondrial innermembrane and is insensitive to feedback inhibition byglucose 6-phosphate This enzyme may monopolize theATP produced in mitochondria, using it to convert glu-cose to glucose 6-phosphate and committing the cell tocontinued glycolysis The hypoxia-inducible transcrip-tion factor (HIF-1) is a protein that acts at the level ofmRNA synthesis to stimulate the synthesis of at leasteight of the glycolytic enzymes This gives the tumorcell the capacity to survive anaerobic conditions untilthe supply of blood vessels has caught up with tumorgrowth

14.1 Glycolysis 533

The German biochemist Otto Warburg was the first

to show, as early as 1928, that tumors have a higher rate

of glucose metabolism than other tissues With his

as-sociates, Warburg purified and crystallized seven of the

enzymes of glycolysis In these studies he developed and

used an experimental tool that revolutionized

biochem-ical studies of oxidative metabolism: the Warburg

manometer, which measured directly the consumption

of oxygen by monitoring changes in gas volume, and

therefore allowed quantitative measurement of any

en-zyme with oxidase activity

Warburg, considered by many the preeminent

bio-chemist of the first half of the twentieth century, made

seminal contributions to manyother areas of biochemistry,including respiration, photo-synthesis, and the enzymol-ogy of intermediary metabo-lism Trained in carbohydratechemistry in the laboratory ofthe great Emil Fischer (whowon the Nobel Prize in Chem-istry in 1902), Warburg him-self won the Nobel Prize inPhysiology or Medicine in

1931 A number of Warburg’sstudents and colleagues alsowere awarded Nobel Prizes:

Otto Meyerhof in 1922, Hans Krebs and Fritz Lipmann

in 1953, and Hugo Theorell in 1955 Meyerhof’s

labora-tory provided training for Lipmann, and for several other

Nobel Prize winners: Severo Ochoa (1959), Andre Lwoff

(1965), and George Wald (1967) ■

■ Glycolysis is a near-universal pathway by which

a glucose molecule is oxidized to two molecules

of pyruvate, with energy conserved as ATP andNADH

■ All ten glycolytic enzymes are in the cytosol,

and all ten intermediates are phosphorylatedcompounds of three or six carbons

■ In the preparatory phase of glycolysis, ATP is

invested to convert glucose to fructose 1,6-bisphosphate The bond between C-3 andC-4 is then broken to yield two molecules oftriose phosphate

■ In the payoff phase, each of the two molecules

of glyceraldehyde 3-phosphate derived fromglucose undergoes oxidation at C-1; the energy

of this oxidation reaction is conserved in theformation of one NADH and two ATP per triosephosphate oxidized The net equation for theoverall process is

Glucose  2NAD   2ADP  2P i88n

2 pyruvate  2NADH  2H   2ATP  2H 2 O

■ Glycolysis is tightly regulated in coordinationwith other energy-yielding pathways to assure

a steady supply of ATP Hexokinase, PFK-1,and pyruvate kinase are all subject to allostericregulation that controls the flow of carbonthrough the pathway and maintains constantlevels of metabolic intermediates

14.2 Feeder Pathways for Glycolysis

Many carbohydrates besides glucose meet their bolic fate in glycolysis, after being transformed into one

cata-of the glycolytic intermediates The most significant arethe storage polysaccharides glycogen and starch; thedisaccharides maltose, lactose, trehalose, and sucrose;and the monosaccharides fructose, mannose, and galac-tose (Fig 14–9)

Glycogen and Starch Are Degraded by Phosphorolysis

Glycogen in animal tissues and in microorganisms (andstarch in plants) can be mobilized for use within thesame cell by a phosphorolytic reaction catalyzed by

glycogen phosphorylase (starch phosphorylase in

plants) These enzymes catalyze an attack by Pion the(1n4) glycosidic linkage that joins the last two glu-

cose residues at a nonreducing end, generating glucose1-phosphate and a polymer one glucose unit shorter

(Fig 14–10) Phosphorolysis preserves some of the

en-ergy of the glycosidic bond in the phosphate ester cose 1-phosphate Glycogen phosphorylase (or starchphosphorylase) acts repetitively until it approaches an(1n6) branch point (see Fig 7–15), where its action

glu-stops A debranching enzyme removes the branches.

The mechanisms and control of glycogen degradationare described in detail in Chapter 15

Glucose 1-phosphate produced by glycogen phorylase is converted to glucose 6-phosphate by

phos-phosphoglucomutase, which catalyzes the reversible

reaction

Glucose 1-phosphate glucose 6-phosphate

The glucose 6-phosphate thus formed can enter ysis or another pathway such as the pentose phosphatepathway, described in Section 14.5 Phosphoglucomu-tase employs essentially the same mechanism as phos-

glycol-phoglycerate mutase (p 531) The general name

mu-tase is given to enzymes that catalyze the transfer of a

functional group from one position to another in the

same molecule Mutases are a subclass of isomerases,

enzymes that interconvert stereoisomers or structural

or positional isomers (see Table 6–3)

Dietary Polysaccharides and Disaccharides Undergo

Hydrolysis to Monosaccharides

For most humans, starch is the major source of

carbo-hydrates in the diet Digestion begins in the mouth,

where salivary
-amylase (Fig 14–9) hydrolyzes the

in-ternal glycosidic linkages of starch, producing short

poly-saccharide fragments or oligopoly-saccharides (Note that in

this hydrolysis reaction, water, not Pi, is the attacking

species.) In the stomach, salivary -amylase is

inacti-vated by the low pH, but a second form of -amylase,

secreted by the pancreas into the small intestine,

con-tinues the breakdown process Pancreatic -amylase

yields mainly maltose and maltotriose (the di- and

trisac-charides of (1n4) glucose) and oligosaccharides called

limit dextrins, fragments of amylopectin containing

(1n6) branch points Maltose and dextrins are

de-graded by enzymes of the intestinal brush border (the

fingerlike microvilli of intestinal epithelial cells, which

greatly increase the area of the intestinal surface)

Di-etary glycogen has essentially the same structure as

starch, and its digestion proceeds by the same pathway

Disaccharides must be hydrolyzed to rides before entering cells Intestinal disaccharides anddextrins are hydrolyzed by enzymes attached to theouter surface of the intestinal epithelial cells:

monosaccha-14.2 Feeder Pathways for Glycolysis 535

O H

H OH

CH2OH

Glyceraldehyde 3-phosphate

sucrase

fructose phosphate aldolase

H H

H

OH HO

Mannose 6-phosphate

Glucose 6-phosphate Sucrose

Trehalose

mutase

D -Galactose

H O

O H H

ATP hexokinase

ATP

ATP

phosphomannose isomerase

Fructose bisphosphate

1,6-triose phosphate isomerase

hexokinase ATP

Fructose 6-phosphate

hexokinase phosphorylase

FIGURE 14–9 Entry of glycogen, starch, disaccharides, and hexoses into the preparatory stage of glycolysis.

trans-Lactose intolerance, common among adults of

most human populations except those originating

in Northern Europe and some parts of Africa, is due to

the disappearance after childhood of most or all of the

lactase activity of the intestinal cells Lactose cannot be

completely digested and absorbed in the small intestine

and passes into the large intestine, where bacteria

con-vert it to toxic products that cause abdominal cramps

and diarrhea The problem is further complicated

be-cause undigested lactose and its metabolites increase

the osmolarity of the intestinal contents, favoring the

retention of water in the intestine In most parts of the

world where lactose intolerance is prevalent, milk is not

used as a food by adults, although milk products

predi-gested with lactase are commercially available in some

countries In certain human disorders, several or all of

the intestinal disaccharidases are missing In these

cases, the digestive disturbances triggered by dietary

disaccharides can sometimes be minimized by a

con-trolled diet ■

Other Monosaccharides Enter the Glycolytic Pathway

at Several Points

In most organisms, hexoses other than glucose can

un-dergo glycolysis after conversion to a phosphorylated

derivative D-Fructose, present in free form in manyfruits and formed by hydrolysis of sucrose in the smallintestine of vertebrates, is phosphorylated by hexokinase:

Mg 2 

Fructose  ATP88nfructose 6-phosphate  ADP

This is a major pathway of fructose entry into sis in the muscles and kidney In the liver, however, fruc-tose enters by a different pathway The liver enzyme

glycoly-fructokinase catalyzes the phosphorylation of fructose

at C-1 rather than C-6:

Mg2

Fructose  ATP88nfructose 1-phosphate  ADP

The fructose 1-phosphate is then cleaved to

glycer-aldehyde and dihydroxyacetone phosphate by fructose

1-phosphate aldolase:

Dihydroxyacetone phosphate is converted to aldehyde 3-phosphate by the glycolytic enzyme triosephosphate isomerase Glyceraldehyde is phosphorylated

glycer-by ATP and triose kinase to glyceraldehyde

3-phos-phate:

Mg 2 

Glyceraldehyde  ATPOn

glyceraldehyde 3-phosphate  ADP

Thus both products of fructose 1-phosphate hydrolysisenter the glycolytic pathway as glyceraldehyde 3-phosphate

D-Galactose, a product of hydrolysis of the accharide lactose (milk sugar), passes in theblood from the intestine to the liver, where it is firstphosphorylated at C-1, at the expense of ATP, by the

dis-enzyme galactokinase:

Mg2

Galactose  ATP88ngalactose 1-phosphate  ADP

The galactose 1-phosphate is then converted to itsepimer at C-4, glucose 1-phosphate, by a set of reac-

tions in which uridine diphosphate (UDP) functions

as a coenzyme-like carrier of hexose groups (Fig.14–11) The epimerization involves first the oxidation ofthe C-4 OOH group to a ketone, then reduction of theketone to an OOH, with inversion of the configuration

at C-4 NAD is the cofactor for both the oxidation andthe reduction

A

A HCOH A

HCOH

CH2OH

A HCOH

Glyceraldehyde

fructose 1-phosphate aldolase

H PO

CH 2 OH Fructose 1-phosphate

Dihydroxyacetone phosphate

C

C

CH 2 OH O

HOCH

C

APA

A 1 2 3 4

P

Glycogen (starch)

n glucose units

glycogen (starch) phosphorylase

H H H

Glycogen (starch)

(n
1) glucose units HO

H H H

CH 2 OH



Glucose 1-phosphate

FIGURE 14–10 Glycogen breakdown by glycogen phosphorylase.

The enzyme catalyzes attack by inorganic phosphate (pink) on the

ter-minal glucosyl residue (blue) at the nonreducing end of a glycogen

molecule, releasing glucose 1-phosphate and generating a glycogen

molecule shortened by one glucose residue The reaction is a

phos-phorolysis (not hydrolysis).

Defects in any of the three enzymes in this pathway

cause galactosemia in humans In

galactokinase-deficiency galactosemia, high galactose concentrations

are found in blood and urine Infants develop cataracts,

caused by deposition of the galactose metabolite

galac-titol in the lens

The symptoms in this disorder are relatively mild, andstrict limitation of galactose in the diet greatly dimin-ishes their severity

Transferase-deficiency galactosemia is more ous; it is characterized by poor growth in children,speech abnormality, mental deficiency, and liver dam-age that may be fatal, even when galactose is withheldfrom the diet Epimerase-deficiency galactosemia leads

seri-to similar sympseri-toms, but is less severe when dietarygalactose is carefully controlled ■

D-Mannose, released in the digestion of various saccharides and glycoproteins of foods, can be phos-phorylated at C-6 by hexokinase:

poly-Mg 2 

Mannose  ATP 88nmannose 6-phosphate  ADP

Mannose 6-phosphate is isomerized by

phosphoman-nose isomerase to yield fructose 6-phosphate, an

in-termediate of glycolysis

SUMMARY 14.2 Feeder Pathways for Glycolysis

■ Glycogen and starch, polymeric storage forms

of glucose, enter glycolysis in a two-stepprocess Phosphorolytic cleavage of a glucoseresidue from an end of the polymer, formingglucose 1-phosphate, is catalyzed by glycogenphosphorylase or starch phosphorylase

Phosphoglucomutase then converts the glucose1-phosphate to glucose 6-phosphate, which canenter glycolysis

■ Ingested polysaccharides and disaccharides areconverted to monosaccharides by intestinalhydrolytic enzymes, and the monosaccharidesthen enter intestinal cells and are transported

to the liver or other tissues

■ A variety of D-hexoses, including fructose,galactose, and mannose, can be funneled intoglycolysis Each is phosphorylated andconverted to either glucose 6-phosphate orfructose 6-phosphate

■ Conversion of galactose 1-phosphate to glucose1-phosphate involves two nucleotide derivatives:UDP-galactose and UDP-glucose Genetic de-fects in any of the three enzymes that catalyzeconversion of galactose to glucose 1-phosphateresult in galactosemias of varying severity

Glucose 1-phosphate

UDP-glucose: galactose phosphate uridylyltransferase

1-Mg2

UDP-glucose

galactokinase

ADP ATP Galactose

glucose

UDP-HO H

H HO

H OH

CH2OH O H OH

Galactose 1-phosphate

UDP-galactose

4 4

UDP

NAD 

NADH  H

UDP-glucose 4-epimerase

NAD 

NADH  H

UDP-glucose 4-epimerase

FIGURE 14–11 Conversion of galactose to glucose 1-phosphate The

conversion proceeds through a sugar-nucleotide derivative,

UDP-galactose, which is formed when galactose 1-phosphate displaces

glu-cose 1-phosphate from UDP-gluglu-cose UDP-galactose is then converted

by UDP-glucose 4-epimerase to UDP-glucose, in a reaction that

in-volves oxidation of C-4 (pink) by NAD, then reduction of C-4 by

NADH; the result is inversion of the configuration at C-4 The

UDP-glucose is recycled through another round of the same reaction The

net effect of this cycle is the conversion of galactose 1-phosphate to

glucose 1-phosphate; there is no net production or consumption of

UDP-galactose or UDP-glucose

14.2 Feeder Pathways for Glycolysis

14.3 Fates of Pyruvate under Anaerobic

Conditions: Fermentation

Pyruvate occupies an important junction in

carbohy-drate catabolism (Fig 14–3) Under aerobic conditions

pyruvate is oxidized to acetate, which enters the citric

acid cycle and is oxidized to CO2and H2O, and NADH

formed by the dehydrogenation of glyceraldehyde

3-phosphate is ultimately reoxidized to NAD
by passage

of its electrons to O2in mitochondrial respiration

How-ever, under hypoxic conditions, as in very active

skele-tal muscle, in submerged plant tissues, or in lactic acid

bacteria, NADH generated by glycolysis cannot be

re-oxidized by O2 Failure to regenerate NAD
would leave

the cell with no electron acceptor for the oxidation of

glyceraldehyde 3-phosphate, and the energy-yielding

reactions of glycolysis would stop NAD
must

there-fore be regenerated in some other way

The earliest cells lived in an atmosphere almost

devoid of oxygen and had to develop strategies for

de-riving energy from fuel molecules under anaerobic

conditions Most modern organisms have retained the

ability to constantly regenerate NAD
during

anaero-bic glycolysis by transferring electrons from NADH to

form a reduced end product such as lactate or ethanol

Pyruvate Is the Terminal Electron Acceptor in Lactic

Acid Fermentation

When animal tissues cannot be supplied with sufficient

oxygen to support aerobic oxidation of the pyruvate and

NADH produced in glycolysis, NAD
is regenerated

from NADH by the reduction of pyruvate to lactate As

mentioned earlier, some tissues and cell types (such as

erythrocytes, which have no mitochondria and thus

can-not oxidize pyruvate to CO2) produce lactate from

glu-cose even under aerobic conditions The reduction of

pyruvate is catalyzed by lactate dehydrogenase,

which forms the Lisomer of lactate at pH 7:

The overall equilibrium of this reaction strongly favors

lactate formation, as shown by the large negative

standard free-energy change

In glycolysis, dehydrogenation of the two molecules

of glyceraldehyde 3-phosphate derived from each

mol-ecule of glucose converts two molmol-ecules of NAD
to two

of NADH Because the reduction of two molecules of

pyruvate to two of lactate regenerates two molecules of

NAD
, there is no net change in NAD
or NADH:

C

CH3Pyruvate

O

lactate dehydrogenase

ery-is produced in large quantities during vigorous musclecontraction (during a sprint, for example), the acidifi-cation that results from ionization of lactic acid in mus-cle and blood limits the period of vigorous activity Thebest-conditioned athletes can sprint at top speed for nomore than a minute (Box 14–1)

Although conversion of glucose to lactate includestwo oxidation-reduction steps, there is no net change inthe oxidation state of carbon; in glucose (C6H12O6) andlactic acid (C3H6O3), the H:C ratio is the same Never-theless, some of the energy of the glucose molecule hasbeen extracted by its conversion to lactate—enough togive a net yield of two molecules of ATP for every glu-

cose molecule consumed Fermentation is the general

term for such processes, which extract energy (as ATP)but do not consume oxygen or change the concentra-tions of NAD
or NADH Fermentations are carried out

by a wide range of organisms, many of which occupyanaerobic niches, and they yield a variety of end prod-ucts, some of which find commercial uses

Ethanol Is the Reduced Product in Ethanol Fermentation

Yeast and other microorganisms ferment glucose toethanol and CO2, rather than to lactate Glucose is con-verted to pyruvate by glycolysis, and the pyruvate isconverted to ethanol and CO2in a two-step process:

In the first step, pyruvate is decarboxylated in an

irre-versible reaction catalyzed by pyruvate

decarboxy-lase This reaction is a simple decarboxylation and does

not involve the net oxidation of pyruvate Pyruvate carboxylase requires Mg2
and has a tightly boundcoenzyme, thiamine pyrophosphate, discussed below

de-In the second step, acetaldehyde is reduced to ethanol

through the action of alcohol dehydrogenase, with

Glucose

2NADH 2NAD

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation 539

Athletes, Alligators, and Coelacanths: Glycolysis

at Limiting Concentrations of Oxygen

Most vertebrates are essentially aerobic organisms;

they convert glucose to pyruvate by glycolysis, thenuse molecular oxygen to oxidize the pyruvate com-pletely to CO2and H2O Anaerobic catabolism of glu-cose to lactate occurs during short bursts of extrememuscular activity, for example in a 100 m sprint, dur-ing which oxygen cannot be carried to the musclesfast enough to oxidize pyruvate Instead, the musclesuse their stored glucose (glycogen) as fuel to gener-ate ATP by fermentation, with lactate as the end prod-uct In a sprint, lactate in the blood builds up to highconcentrations It is slowly converted back to glucose

by gluconeogenesis in the liver in the subsequent rest

or recovery period, during which oxygen is consumed

at a gradually diminishing rate until the breathing ratereturns to normal The excess oxygen consumed inthe recovery period represents a repayment of theoxygen debt This is the amount of oxygen required

to supply ATP for gluconeogenesis during recoveryrespiration, in order to regenerate the glycogen “bor-rowed” from liver and muscle to carry out intense mus-cular activity in the sprint The cycle of reactions thatincludes glucose conversion to lactate in muscle andlactate conversion to glucose in liver is called the Coricycle, for Carl and Gerty Cori, whose studies in the1930s and 1940s clarified the pathway and its role (seeBox 15–1)

The circulatory systems of most small vertebratescan carry oxygen to their muscles fast enough to avoidhaving to use muscle glycogen anaerobically For ex-ample, migrating birds often fly great distances at highspeeds without rest and without incurring an oxygendebt Many running animals of moderate size also main-tain an essentially aerobic metabolism in their skele-tal muscle However, the circulatory systems of largeranimals, including humans, cannot completely sustainaerobic metabolism in skeletal muscles over long pe-riods of intense muscular activity These animals gen-erally are slow-moving under normal circumstances andengage in intense muscular activity only in the gravestemergencies, because such bursts of activity requirelong recovery periods to repay the oxygen debt

Alligators and crocodiles, for example, are mally sluggish animals Yet when provoked they arecapable of lightning-fast charges and dangerous lash-ings of their powerful tails Such intense bursts of ac-tivity are short and must be followed by long periods

nor-of recovery The fast emergency movements require

lactic acid fermentation to generate ATP in skeletalmuscles The stores of muscle glycogen are rapidly ex-pended in intense muscular activity, and lactatereaches very high concentrations in muscles and ex-tracellular fluid Whereas a trained athlete can recoverfrom a 100 m sprint in 30 min or less, an alligator mayrequire many hours of rest and extra oxygen con-sumption to clear the excess lactate from its blood andregenerate muscle glycogen after a burst of activity.Other large animals, such as the elephant and rhi-noceros, have similar metabolic characteristics, as dodiving mammals such as whales and seals Dinosaursand other huge, now-extinct animals probably had todepend on lactic acid fermentation to supply energyfor muscular activity, followed by very long recoveryperiods during which they were vulnerable to attack

by smaller predators better able to use oxygen andthus better adapted to continuous, sustained muscu-lar activity

Deep-sea explorations have revealed manyspecies of marine life at great ocean depths, where theoxygen concentration is near zero For example, theprimitive coelacanth, a large fish recovered fromdepths of 4,000 m or more off the coast of SouthAfrica, has an essentially anaerobic metabolism in vir-tually all its tissues It converts carbohydrates to lac-tate and other products, most of which must be ex-creted Some marine vertebrates ferment glucose toethanol and CO2in order to generate ATP

the reducing power furnished by NADH derived from

the dehydrogenation of glyceraldehyde 3-phosphate

This reaction is a well-studied case of hydride transfer

from NADH (Fig 14–12) Ethanol and CO2are thus the

end products of ethanol fermentation, and the overall

equation is

Glucose  2ADP  2P i88n

2 ethanol  2CO 2  2ATP  2H 2 O

As in lactic acid fermentation, there is no net change in

the ratio of hydrogen to carbon atoms when glucose

(H:C ratio 12/6  2) is fermented to two ethanol and

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

540

N

H H

C O

NH2NADH

H

N+R

C O

NH2 +

C O Zn 2+

C H

H OH

CH3

H

Alcohol dehydrogenase

MECHANISM FIGURE 14–12 The alcohol dehydrogenase reaction.

A Zn2at the active site polarizes the carbonyl oxygen of acetaldehyde,

allowing transfer of a hydride ion (red) from the reduced cofactor

NADH The reduced intermediate acquires a proton from the medium

(blue) to form ethanol. Alcohol Dehydrogenase Mechanism

TABLE 14–1 Some TPP-Dependent Reactions

Pyruvate decarboxylase Ethanol fermentation

Pyruvate dehydrogenase Synthesis of acetyl-CoA

-Ketoglutarate dehydrogenase Citric acid cycle

Transketolase Carbon-assimilation reactions

O

R 2 C C

O O

O

R 1 C H

O

R 1 C C

O O

fer-Pyruvate decarboxylase is present in brewer’s andbaker’s yeast and in all other organisms that fermentglucose to ethanol, including some plants The CO2pro-duced by pyruvate decarboxylation in brewer’s yeast isresponsible for the characteristic carbonation of cham-pagne The ancient art of brewing beer involves a num-ber of enzymatic processes in addition to the reactions

of ethanol fermentation (Box 14–2) In baking, CO2leased by pyruvate decarboxylase when yeast is mixedwith a fermentable sugar causes dough to rise The en-zyme is absent in vertebrate tissues and in other or-ganisms that carry out lactic acid fermentation Alcohol dehydrogenase is present in many organ-isms that metabolize ethanol, including humans In hu-man liver it catalyzes the oxidation of ethanol, either in-gested or produced by intestinal microorganisms, withthe concomitant reduction of NADto NADH

re-Thiamine Pyrophosphate Carries

“Active Acetaldehyde” Groups

The pyruvate decarboxylase reaction provides our first

encounter with thiamine pyrophosphate (TPP) (Fig.

14–13), a coenzyme derived from vitamin B1 Lack of tamin B1in the human diet leads to the condition known

vi-as beriberi, characterized by an accumulation of bodyfluids (swelling), pain, paralysis, and ultimately death.Thiamine pyrophosphate plays an important role inthe cleavage of bonds adjacent to a carbonyl group, such

as the decarboxylation of -keto acids, and in chemical

rearrangements in which an activated acetaldehydegroup is transferred from one carbon atom to another(Table 14–1) The functional part of TPP, the thiazoliumring, has a relatively acidic proton at C-2 Loss of this

active acetaldehyde CH3

OH

O

C C

CH 3

NH 2

CH 2

N N

H

CH3

5 4

C

O N

CH3S

R

Acetaldehyde

R

resonance stabilization

CH3 C

H O

H

CO2

O OH C

C N

CH3

S

R R

C N

CH 3

S

R R

C N

5 R

CH3

S

R R

C H

N

CH3S

proton produces a carbanion that is the active species

in TPP-dependent reactions (Fig 14–13) The

carban-ion readily adds to carbonyl groups, and the thiazolium

ring is thereby positioned to act as an “electron sink”

that greatly facilitates reactions such as the

decarboxy-lation catalyzed by pyruvate decarboxylase

Fermentations Yield a Variety of Common Foods and

Industrial Chemicals

Our progenitors learned millennia ago to use

fermenta-tion in the producfermenta-tion and preservafermenta-tion of foods

Cer-tain microorganisms present in raw food products

fer-ment the carbohydrates and yield metabolic productsthat give the foods their characteristic forms, textures,and tastes Yogurt, already known in Biblical times, is

produced when the bacterium Lactobacillus cus ferments the carbohydrate in milk, producing lac-

bulgari-tic acid; the resulting drop in pH causes the milk teins to precipitate, producing the thick texture andsour taste of unsweetened yogurt Another bacterium,

pro-Propionibacterium freudenreichii, ferments milk to

produce propionic acid and CO2; the propionic acid cipitates milk proteins, and bubbles of CO2 cause theholes characteristic of Swiss cheese Many other foodproducts are the result of fermentations: pickles, sauer-kraut, sausage, soy sauce, and a variety of national fa-vorites, such as kimchi (Korea), tempoyak (Indonesia),kefir (Russia), dahi (India), and pozol (Mexico) Thedrop in pH associated with fermentation also helps topreserve foods, because most of the microorganismsthat cause food spoilage cannot grow at low pH Inagriculture, plant byproducts such as corn stalks arepreserved for use as animal feed by packing them into

pre-a lpre-arge contpre-ainer (pre-a silo) with limited pre-access to pre-air;microbial fermentation produces acids that lower the

pH The silage that results from this fermentation

14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation 541

MECHANISM FIGURE 14–13 Thiamine pyrophosphate (TPP) and its

role in pyruvate decarboxylation (a) TPP is the coenzyme form of

vi-tamin B 1 (thiamine) The reactive carbon atom in the thiazolium ring

of TPP is shown in red In the reaction catalyzed by pyruvate

decar-boxylase, two of the three carbons of pyruvate are carried transiently

on TPP in the form of a hydroxyethyl, or “active acetaldehyde,” group

(b), which is subsequently released as acetaldehyde (c) After cleavage

of a carbon–carbon bond, one product often has a free electron pair,

or carbanion, which because of its strong tendency to form a new bond

is generally unstable The thiazolium ring of TPP stabilizes carbanion

intermediates by providing an electrophilic (electron-deficient)

struc-ture into which the carbanion electrons can be delocalized by

reso-nance Structures with this property, often called “electron sinks,” play

a role in many biochemical reactions This principle is illustrated here

for the reaction catalyzed by pyruvate decarboxylase 1 The TPP

car-banion acts as a nucleophile, attacking the carbonyl group of pyruvate.

2 Decarboxylation produces a carbanion that is stabilized by the

thiazolium ring 3 Protonation to form hydroxyethyl TPP is followed

by 4 release of acetaldehyde 5 A proton dissociates to regenerate

the carbanion. Thiamine Pyrophosphate Mechanism

process can be kept as animal feed for long periods

without spoilage

In 1910 Chaim Weizmann (later to become the first

president of Israel) discovered that the bacterium

Clostridium acetobutyricum ferments starch to

bu-tanol and acetone This discovery opened the field of

industrial fermentations, in which some readily

avail-able material rich in carbohydrate (corn starch or

mo-lasses, for example) is supplied to a pure culture of a

specific microorganism, which ferments it into a

prod-uct of greater value The methanol used to make

“gaso-hol” is produced by microbial fermentation, as are

formic, acetic, propionic, butyric, and succinic acids,

and glycerol, ethanol, isopropanol, butanol, and

bu-tanediol These fermentations are generally carried out

in huge closed vats in which temperature and access to

air are adjusted to favor the multiplication of the

de-sired microorganism and to exclude contaminating

organisms (Fig 14–14) The beauty of industrial

fer-mentations is that complicated, multistep chemical

transformations are carried out in high yields and with

few side products by chemical factories that reproduce

themselves—microbial cells For some industrial

fer-mentations, technology has been developed to

immobi-lize the cells in an inert support, to pass the starting

ma-terial continuously through the bed of immobilized cells,

and to collect the desired product in the effluent—an

engineer’s dream!

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

542

FIGURE 14–14 Industrial-scale fermentation Microorganisms are

cultured in a sterilizable vessel containing thousands of liters of growth medium—an inexpensive source of both carbon and energy—under carefully controlled conditions, including low oxygen concentration and constant temperature After centrifugal separation of the cells from the growth medium, the valuable products of the fermentation are re- covered from the cells or from the supernatant fluid.

Brewing Beer

Brewers prepare beer by ethanol fermentation of the

carbohydrates in cereal grains (seeds) such as barley,

carried out by yeast glycolytic enzymes The

carbo-hydrates, largely polysaccharides, must first be

de-graded to disaccharides and monosaccharides In a

process called malting, the barley seeds are allowed

to germinate until they form the hydrolytic enzymes

required to break down their polysaccharides, at

which point germination is stopped by controlled

heat-ing The product is malt, which contains enzymes that

catalyze the hydrolysis of the  linkages of cellulose

and other cell wall polysaccharides of the barley husks,

and enzymes such as -amylase and maltase.

The brewer next prepares the wort, the nutrientmedium required for fermentation by yeast cells The

malt is mixed with water and then mashed or crushed

This allows the enzymes formed in the malting process

to act on the cereal polysaccharides to form maltose,

glucose, and other simple sugars, which are soluble in

the aqueous medium The remaining cell matter is

then separated, and the liquid wort is boiled with hops

to give flavor The wort is cooled and then aerated

Now the yeast cells are added In the aerobic wortthe yeast grows and reproduces very rapidly, using en-ergy obtained from available sugars No ethanol formsduring this stage, because the yeast, amply suppliedwith oxygen, oxidizes the pyruvate formed by glycoly-sis to CO2and H2O via the citric acid cycle When allthe dissolved oxygen in the vat of wort has been con-sumed, the yeast cells switch to anaerobic metabolism,and from this point they ferment the sugars into ethanoland CO2 The fermentation process is controlled in part

by the concentration of the ethanol formed, by the pH,and by the amount of remaining sugar After fermen-tation has been stopped, the cells are removed and the

“raw” beer is ready for final processing

In the final steps of brewing, the amount of foam

or head on the beer, which results from dissolved teins, is adjusted Normally this is controlled by pro-teolytic enzymes that arise in the malting process Ifthese enzymes act on the proteins too long, the beerwill have very little head and will be flat; if they donot act long enough, the beer will not be clear when

pro-it is cold Sometimes proteolytic enzymes from othersources are added to control the head

SUMMARY 14.3 Fates of Pyruvate under Anaerobic

Conditions: Fermentation

■ The NADH formed in glycolysis must berecycled to regenerate NAD, which isrequired as an electron acceptor in the firststep of the payoff phase Under aerobicconditions, electrons pass from NADH to O2inmitochondrial respiration

■ Under anaerobic or hypoxic conditions, manyorganisms regenerate NADby transferringelectrons from NADH to pyruvate, forminglactate Other organisms, such as yeast,regenerate NAD by reducing pyruvate toethanol and CO2 In these anaerobic processes

(fermentations), there is no net oxidation or

reduction of the carbons of glucose

■ A variety of microorganisms can ferment sugar

in fresh foods, resulting in changes in pH, taste,and texture, and preserving food from spoilage

Fermentations are used in industry to produce

a wide variety of commercially valuable organiccompounds from inexpensive starting materials

14.4 Gluconeogenesis

The central role of glucose in metabolism arose early in

evolution, and this sugar remains the nearly universal

fuel and building block in modern organisms, from

mi-crobes to humans In mammals, some tissues depend

almost completely on glucose for their metabolic energy

For the human brain and nervous system, as well as the

erythrocytes, testes, renal medulla, and embryonic

tis-sues, glucose from the blood is the sole or major fuel

source The brain alone requires about 120 g of glucose

each day—more than half of all the glucose stored as

glycogen in muscle and liver However, the supply of

glu-cose from these stores is not always sufficient; between

meals and during longer fasts, or after vigorous

exer-cise, glycogen is depleted For these times, organisms

need a method for synthesizing glucose from

noncar-bohydrate precursors This is accomplished by a

path-way called gluconeogenesis (“formation of new

sugar”), which converts pyruvate and related three- and

four-carbon compounds to glucose

Gluconeogenesis occurs in all animals, plants, fungi,and microorganisms The reactions are essentially the

same in all tissues and all species The important

pre-cursors of glucose in animals are three-carbon

com-pounds such as lactate, pyruvate, and glycerol, as well

as certain amino acids (Fig 14–15) In mammals,

glu-coneogenesis takes place mainly in the liver, and to a

lesser extent in renal cortex The glucose produced

passes into the blood to supply other tissues After

vig-orous exercise, lactate produced by anaerobic

glycoly-sis in skeletal muscle returns to the liver and is verted to glucose, which moves back to muscle and isconverted to glycogen—a circuit called the Cori cycle(Box 14–1; see also Fig 23–18) In plant seedlings,stored fats and proteins are converted, via paths thatinclude gluconeogenesis, to the disaccharide sucrose fortransport throughout the developing plant Glucose andits derivatives are precursors for the synthesis of plantcell walls, nucleotides and coenzymes, and a variety ofother essential metabolites In many microorganisms,gluconeogenesis starts from simple organic compounds

con-of two or three carbons, such as acetate, lactate, andpropionate, in their growth medium

Although the reactions of gluconeogenesis are thesame in all organisms, the metabolic context and theregulation of the pathway differ from one species to an-other and from tissue to tissue In this section we focus

on gluconeogenesis as it occurs in the mammalian liver

In Chapter 20 we show how photosynthetic organismsuse this pathway to convert the primary products ofphotosynthesis into glucose, to be stored as sucrose orstarch

14.4 Gluconeogenesis 543

Glycoproteins

Blood glucose Glycogen

Glucogenic amino acids

Citric acid cycle

Glucose 6-phosphate

Other monosaccharides Sucrose Disaccharides

Pyruvate

Lactate

pyruvate

Phosphoenol- glycerate

3-Phospho-CO2fixation

glycerols Glycerol

Starch

Energy

FIGURE 14–15 Carbohydrate synthesis from simple precursors The

pathway from phosphoenolpyruvate to glucose 6-phosphate is mon to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants Plants and photosynthetic bac- teria are uniquely able to convert CO to carbohydrates.

com-Gluconeogenesis and glycolysis are not identical

pathways running in opposite directions, although they

do share several steps (Fig 14–16); seven of the ten

en-zymatic reactions of gluconeogenesis are the reverse of

glycolytic reactions However, three reactions of

glycol-ysis are essentially irreversible in vivo and cannot be

used in gluconeogenesis: the conversion of glucose to

glucose 6-phosphate by hexokinase, the

phosphoryla-tion of fructose 6-phosphate to fructose

1,6-bisphos-phate by phosphofructokinase-1, and the conversion of

phosphoenolpyruvate to pyruvate by pyruvate kinase

(Fig 14–16) In cells, these three reactions are

charac-terized by a large negative free-energy change, G,

whereas other glycolytic reactions have a G near 0

(Table 14–2) In gluconeogenesis, the three irreversiblesteps are bypassed by a separate set of enzymes, cat-alyzing reactions that are sufficiently exergonic to be ef-fectively irreversible in the direction of glucose synthe-sis Thus, both glycolysis and gluconeogenesis areirreversible processes in cells In animals, both pathwaysoccur largely in the cytosol, necessitating their recipro-cal and coordinated regulation Separate regulation ofthe two pathways is brought about through controls ex-erted on the enzymatic steps unique to each

We begin by considering the three bypass reactions

of gluconeogenesis (Keep in mind that “bypass” refersthroughout to the bypass of irreversible glycolytic re-actions.)

Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions

The first of the bypass reactions in gluconeogenesis isthe conversion of pyruvate to phosphoenolpyruvate(PEP) This reaction cannot occur by reversal of thepyruvate kinase reaction of glycolysis (p 532), whichhas a large, negative standard free-energy change and

is irreversible under the conditions prevailing in intactcells (Table 14–2, step 10) Instead, the phosphoryla-tion of pyruvate is achieved by a roundabout sequence

of reactions that in eukaryotes requires enzymes in boththe cytosol and mitochondria As we shall see, the path-way shown in Figure 14–16 and described in detail here

is one of two routes from pyruvate to PEP; it is the dominant path when pyruvate or alanine is the gluco-genic precursor A second pathway, described later, pre-dominates when lactate is the glucogenic precursor.Pyruvate is first transported from the cytosol intomitochondria or is generated from alanine within mito-chondria by transamination, in which the -amino group

pre-is removed from alanine (leaving pyruvate) and added

to an -keto carboxylic acid (transamination reactions

are discussed in detail in Chapter 18) Then pyruvate

carboxylase, a mitochondrial enzyme that requires the

coenzyme biotin, converts the pyruvate to oxaloacetate

(2) ATP

(2) ADP

(2) ATP

(2) ATP (2) ADP

Dihydroxyacetone phosphate

Dihydroxyacetone

phosphate

(2) Glyceraldehyde 3-phosphate

(2) NAD(2) NAD

FIGURE 14–16 Opposing pathways of glycolysis and sis in rat liver The reactions of glycolysis are shown on the left side

gluconeogene-in blue; the opposgluconeogene-ing pathway of gluconeogenesis is shown on the right in red The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15 Figure 14–19 illustrates an alternative route for oxaloacetate produced

in mitochondria.

The reaction involves biotin as a carrier of activated

HCO3
(Fig 14–18) The reaction mechanism is shown

in Figure 16–16 Pyruvate carboxylase is the first

regu-latory enzyme in the gluconeogenic pathway, requiring

acetyl-CoA as a positive effector (Acetyl-CoA is

pro-duced by fatty acid oxidation (Chapter 17), and its

ac-cumulation signals the availability of fatty acids as fuel.)

As we shall see in Chapter 16 (see Fig 16–15), the

pyru-vate carboxylase reaction can replenish intermediates

in another central metabolic pathway, the citric acid

cycle

Because the mitochondrial membrane has no porter for oxaloacetate, before export to the cytosol the

trans-oxaloacetate formed from pyruvate must be reduced to

malate by mitochondrial malate dehydrogenase, at

the expense of NADH:

Oxaloacetate  NADH  H  z L-malate  NAD  (14–5)

14.4 Gluconeogenesis 545

TABLE 14–2 Free-Energy Changes of Glycolytic Reactions in Erythrocytes

4 Fructose 1,6-bisphosphate dihydroxyacetone phosphate

6 Glyceraldehyde 3-phosphate Pi NAD 1,3-bisphosphoglycerate 

z

zz

zz

Note: G is the standard free-energy change, as defined in Chapter 13 (p 491) G is the free-energy change calculated from the actual

concentrations of glycolytic intermediates present under physiological conditions in erythrocytes, at pH 7 The glycolytic reactions bypassed

in gluconeogenesis are shown in red Biochemical equations are not necessarily balanced for H or charge (p 506).



O

Guanosine

PEP carboxykinase

Pyruvate

biotin pyruvate carboxylase

O
O

O

Bicarbonate

O C

FIGURE 14–17 Synthesis of phosphoenolpyruvate from pyruvate.

(a) In mitochondria, pyruvate is converted to oxaloacetate in a

biotin-requiring reaction catalyzed by pyruvate carboxylase (b) In the cytosol,

oxaloacetate is converted to phosphoenolpyruvate by PEP

carboxy-kinase The CO 2 incorporated in the pyruvate carboxylase reaction is

lost here as CO 2 The decarboxylation leads to a rearrangement of

electrons that facilitates attack of the carbonyl oxygen of the pyruvate

moiety on the
phosphate of GTP.

The standard free-energy change for this reaction isquite high, but under physiological conditions (includ-ing a very low concentration of oxaloacetate)
G ≈ 0 and

the reaction is readily reversible Mitochondrial malatedehydrogenase functions in both gluconeogenesis andthe citric acid cycle, but the overall flow of metabolites

in the two processes is in opposite directions

Malate leaves the mitochondrion through a specifictransporter in the inner mitochondrial membrane (seeFig 19–27), and in the cytosol it is reoxidized to ox-aloacetate, with the production of cytosolic NADH:

Malate  NAD 88noxaloacetate  NADH  H  (14–6)

The oxaloacetate is then converted to PEP by

phosphoenolpyruvate carboxykinase (Fig 14–17).

This Mg2-dependent reaction requires GTP as thephosphoryl group donor :

Oxaloacetate  GTP PEP  CO 2  GDP (14–7)

The reaction is reversible under intracellular conditions;the formation of one high-energy phosphate compound(PEP) is balanced by the hydrolysis of another (GTP).The overall equation for this set of bypass reactions,the sum of Equations 14–4 through 14–7, is

Pyruvate  ATP  GTP  HCO 3 88n

PEP  ADP  GDP  P i  CO 2

G  0.9 kJ/mol (14–8)

Two high-energy phosphate equivalents (one from ATPand one from GTP), each yielding about 50 kJ/mol un-der cellular conditions, must be expended to phosphor-ylate one molecule of pyruvate to PEP In contrast, whenPEP is converted to pyruvate during glycolysis, only oneATP is generated from ADP Although the standard free-energy change (
G) of the two-step path from pyru-

vate to PEP is 0.9 kJ/mol, the actual free-energy change(
G), calculated from measured cellular concentrations

of intermediates, is very strongly negative (25 kJ/mol);this results from the ready consumption of PEP in otherreactions such that its concentration remains relativelylow The reaction is thus effectively irreversible in thecell

Note that the CO2added to pyruvate in the vate carboxylase step is the same molecule that is lost

pyru-in the PEP carboxykpyru-inase reaction (Fig 14–17) Thiscarboxylation-decarboxylation sequence represents away of “activating” pyruvate, in that the decarboxyla-tion of oxaloacetate facilitates PEP formation In Chap-ter 21 we shall see how a similar carboxylation-decar-boxylation sequence is used to activate acetyl-CoA forfatty acid biosynthesis (see Fig 21–1)

There is a logic to the route of these reactionsthrough the mitochondrion The [NADH]/[NAD] ratio

in the cytosol is 8 104, about 105

times lower than

in mitochondria Because cytosolic NADH is consumed

in gluconeogenesis (in the conversion of

1,3-bisphos-z

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

546

O O P

ATP Rib Adenine

Enz

O

OH

O O P

O

O

C

OO

C

OO

O

O

C O

O O P

N H Enz

FIGURE 14–18 Role of biotin in the pyruvate carboxylase reaction.

The cofactor biotin is covalently attached to the enzyme through an

amide linkage to the
-amino group of a Lys residue, forming a

biotinyl-enzyme The reaction occurs in two phases, which occur at

two different sites in the enzyme At catalytic site 1, bicarbonate ion

is converted to CO 2 at the expense of ATP Then CO 2 reacts with

biotin, forming carboxybiotinyl-enzyme The long arm composed of

biotin and the side chain of the Lys to which it is attached then carry

the CO 2 of carboxybiotinyl-enzyme to catalytic site 2 on the enzyme

surface, where CO 2 is released and reacts with the pyruvate, forming

oxaloacetate and regenerating the biotinyl-enzyme The general role

of flexible arms in carrying reaction intermediates between enzyme

active sites is described in Figure 16–17, and the mechanistic details

of the pyruvate carboxylase reaction are shown in Figure 16–16

Sim-ilar mechanisms occur in other biotin-dependent carboxylation

reac-tions, such as those catalyzed by propionyl-CoA carboxylase (see Fig.

17–11) and acetyl-CoA carboxylase (see Fig 21–1).

phoglycerate to glyceraldehyde 3-phosphate; Fig.

14–16), glucose biosynthesis cannot proceed unless

NADH is available The transport of malate from the

mi-tochondrion to the cytosol and its reconversion there to

oxaloacetate effectively moves reducing equivalents to

the cytosol, where they are scarce This path from

pyru-vate to PEP therefore provides an important balance

be-tween NADH produced and consumed in the cytosol

during gluconeogenesis

A second pyruvate n PEP bypass predominateswhen lactate is the glucogenic precursor (Fig 14–19)

This pathway makes use of lactate produced by

glycol-ysis in erythrocytes or anaerobic muscle, for example,

and it is particularly important in large vertebrates

af-ter vigorous exercise (Box 14–1) The conversion of

lac-tate to pyruvate in the cytosol of hepatocytes yields

NADH, and the export of reducing equivalents (as

malate) from mitochondria is therefore unnecessary

Af-ter the pyruvate produced by the lactate dehydrogenase

reaction is transported into the mitochondrion, it is

con-verted to oxaloacetate by pyruvate carboxylase, as

de-scribed above This oxaloacetate, however, is converted

directly to PEP by a mitochondrial isozyme of PEP

car-boxykinase, and the PEP is transported out of the

mi-tochondrion to continue on the gluconeogenic path The

mitochondrial and cytosolic isozymes of PEP

carboxy-kinase are encoded by separate genes in the nuclear

chromosomes, providing another example of two

dis-tinct enzymes catalyzing the same reaction but having

different cellular locations or metabolic roles (recall the

isozymes of hexokinase)

Conversion of Fructose 1,6-Bisphosphate to

Fructose 6-Phosphate Is the Second Bypass

The second glycolytic reaction that cannot participate

in gluconeogenesis is the phosphorylation of fructose

6-phosphate by PFK-1 (Table 14–2, step 3 ) Because this

reaction is highly exergonic and therefore irreversible

in intact cells, the generation of fructose 6-phosphate

from fructose 1,6-bisphosphate (Fig 14–16) is catalyzed

by a different enzyme, Mg2 -dependent fructose

1,6-bisphosphatase (FBPase-1), which promotes the

es-sentially irreversible hydrolysis of the C-1 phosphate

(not phosphoryl group transfer to ADP):

Fructose 1,6-bisphosphate  H 2 O88n

fructose 6-phosphate  P i

G 
16.3 kJ/mol

Conversion of Glucose 6-Phosphate to Glucose

Is the Third Bypass

The third bypass is the final reaction of

gluconeogene-sis, the dephosphorylation of glucose 6-phosphate to

yield glucose (Fig 14–16) Reversal of the hexokinase

reaction (p 526) would require phosphoryl group

trans-fer from glucose 6-phosphate to ADP, forming ATP, anenergetically unfavorable reaction (Table 14–2, step 1

) The reaction catalyzed by glucose 6-phosphatase

does not require synthesis of ATP; it is a simple drolysis of a phosphate ester:

hy-Glucose 6-phosphate  H 2 OOnglucose  P i

G 
13.8 kJ/mol

This Mg2-activated enzyme is found on the lumenalside of the endoplasmic reticulum of hepatocytes andrenal cells (see Fig 15–6) Muscle and brain tissue donot contain this enzyme and so cannot carry out gluco-neogenesis Glucose produced by gluconeogenesis inthe liver or kidney or ingested in the diet is delivered

to brain and muscle through the bloodstream

14.4 Gluconeogenesis 547

cytosolic malate dehydrogenase

mitochondrial malate dehydrogenase

Pyruvate Pyruvate Oxaloacetate

Malate Malate

Oxaloacetate

cytosolic PEP carboxykinase

CO2PEP

CO2Oxaloacetate

Pyruvate

Lactate

PEP

mitochondrial PEP carboxykinase

CO2

pyruvate carboxylase

NAD+

lactate dehydrogenase

Mitochondrion Cytosol

Pyruvate

pyruvate carboxylase

NADH + H+

NAD+NADH + H+

NAD+NADH + H+

shut-Gluconeogenesis Is Energetically Expensive,

but Essential

The sum of the biosynthetic reactions leading from

pyruvate to free blood glucose (Table 14–3) is

2 Pyruvate  4ATP  2GTP  2NADH  2H   4H 2 O88n

glucose  4ADP  2GDP  6P i  2NAD  (14–9)

For each molecule of glucose formed from pyruvate, six

high-energy phosphate groups are required, four from

ATP and two from GTP In addition, two molecules of

NADH are required for the reduction of two molecules

of 1,3-bisphosphoglycerate Clearly, Equation 14–9 is

not simply the reverse of the equation for conversion of

glucose to pyruvate by glycolysis, which requires only

two molecules of ATP:

Glucose  2ADP  2P i  2NAD 88n

2 pyruvate  2ATP  2NADH  2H   2H 2 O

The synthesis of glucose from pyruvate is a relatively

expensive process Much of this high energy cost is

nec-essary to ensure the irreversibility of gluconeogenesis

Under intracellular conditions, the overall free-energy

change of glycolysis is at least
63 kJ/mol Under the

same conditions the overall G of gluconeogenesis is

16 kJ/mol Thus both glycolysis and gluconeogenesis

are essentially irreversible processes in cells

Citric Acid Cycle Intermediates and Many Amino

Acids Are Glucogenic

The biosynthetic pathway to glucose described above

allows the net synthesis of glucose not only from

pyru-vate but also from the four-, five-, and six-carbon

inter-mediates of the citric acid cycle (Chapter 16) Citrate,

isocitrate, -ketoglutarate, succinyl-CoA, succinate,

fu-marate, and malate—all are citric acid cycle ates that can undergo oxidation to oxaloacetate (seeFig 16–7) Some or all of the carbon atoms of mostamino acids derived from proteins are ultimately catab-olized to pyruvate or to intermediates of the citric acidcycle Such amino acids can therefore undergo net con-

intermedi-version to glucose and are said to be glucogenic (Table

14–4) Alanine and glutamine, the principal moleculesthat transport amino groups from extrahepatic tissues

to the liver (see Fig 18–9), are particularly importantglucogenic amino acids in mammals After removal oftheir amino groups in liver mitochondria, the carbonskeletons remaining (pyruvate and -ketoglutarate, re-

spectively) are readily funneled into gluconeogenesis

In contrast, no net conversion of fatty acids to cose occurs in mammals As we shall see in Chapter 17,the catabolism of most fatty acids yields only acetyl-CoA Mammals cannot use acetyl-CoA as a precursor ofglucose, because the pyruvate dehydrogenase reaction

glu-is irreversible and cells have no other pathway to vert acetyl-CoA to pyruvate Plants, yeast, and manybacteria do have a pathway (the glyoxylate cycle; seeFig 16–20) for converting acetyl-CoA to oxaloacetate,

con-so these organisms can use fatty acids as the startingmaterial for gluconeogenesis This is especially impor-tant during the germination of seedlings, before photo-synthesis can serve as a source of glucose

Glycolysis and Gluconeogenesis Are Regulated Reciprocally

If glycolysis (the conversion of glucose to pyruvate) andgluconeogenesis (the conversion of pyruvate to glucose)were allowed to proceed simultaneously at high rates,

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

548

TABLE 14–3 Sequential Reactions in Gluconeogenesis Starting from Pyruvate

1,3-Bisphosphoglycerate NADH  H glyceraldehyde 3-phosphate NAD Pi 2Glyceraldehyde 3-phosphate dihydroxyacetone phosphate

Glyceraldehyde 3-phosphate dihydroxyacetone phosphate fructose 1,6-bisphosphate

Fructose 1,6-bisphosphate On fructose 6-phosphate  Pi

Fructose 6-phosphate glucose 6-phosphate

Glucose 6-phosphate H2O On glucose  Pi

Sum: 2 Pyruvate 4ATP  2GTP  2NADH  2H 4H2O On glucose  4ADP  2GDP  6Pi 2NAD

z

zz

zz

z

zz

Note: The bypass reactions are in red; all other reactions are reversible steps of glycolysis The figures at the right indicate that the reaction is to be counted twice,

because two three-carbon precursors are required to make a molecule of glucose The reactions required to replace the cytosolic NADH consumed in the

glycer-aldehyde 3-phosphate dehydrogenase reaction (the conversion of lactate to pyruvate in the cytosol or the transport of reducing equivalents from mitochondria to

the result would be the consumption of ATP and the

production of heat For example, PFK-1 and FBPase-1

catalyze opposing reactions:

ATP  fructose 6-phosphate8888888n

ATP  H 2 O88nADP  P i  heat

These two enzymatic reactions, and a number of others

in the two pathways, are regulated allosterically and by

covalent modification (phosphorylation) In Chapter 15

we take up the mechanisms of this regulation in detail

For now, suffice it to say that the pathways are

regu-lated so that when the flux of glucose through

glycoly-sis goes up, the flux of pyruvate toward glucose goes

down, and vice versa

■ Gluconeogenesis is a ubiquitous multistepprocess in which pyruvate or a related three-carbon compound (lactate, alanine) isconverted to glucose Seven of the steps ingluconeogenesis are catalyzed by the sameenzymes used in glycolysis; these are thereversible reactions

■ Three irreversible steps in the glycolyticpathway are bypassed by reactions catalyzed

by gluconeogenic enzymes: (1) conversion of

pyruvate to PEP via oxaloacetate, catalyzed bypyruvate carboxylase and PEP carboxykinase;(2) dephosphorylation of fructose

1,6-bisphosphate by FBPase-1; and (3) dephosphorylation of glucose 6-phosphate

■ Pyruvate carboxylase is stimulated by acetyl-CoA, increasing the rate ofgluconeogenesis when the cell already hasadequate supplies of other substrates (fattyacids) for energy production

■ Animals cannot convert acetyl-CoA derivedfrom fatty acids into glucose; plants andmicroorganisms can

■ Glycolysis and gluconeogenesis are reciprocallyregulated to prevent wasteful operation of bothpathways at the same time

14.5 Pentose Phosphate Pathway of Glucose Oxidation

In most animal tissues, the major catabolic fate

of glucose 6-phosphate is glycolytic breakdown

to pyruvate, much of which is then oxidized via thecitric acid cycle, ultimately leading to the formation ofATP Glucose 6-phosphate does have other catabolicfates, however, which lead to specialized productsneeded by the cell Of particular importance in sometissues is the oxidation of glucose 6-phosphate to pen-

tose phosphates by the pentose phosphate pathway (also called the phosphogluconate pathway or the

hexose monophosphate pathway; Fig 14–20) In this

oxidative pathway, NADP is the electron acceptor,yielding NADPH Rapidly dividing cells, such as those ofbone marrow, skin, and intestinal mucosa, use the pen-toses to make RNA, DNA, and such coenzymes as ATP,NADH, FADH2, and coenzyme A

In other tissues, the essential product of the tose phosphate pathway is not the pentoses but the elec-tron donor NADPH, needed for reductive biosynthesis

pen-or to counter the damaging effects of oxygen radicals.Tissues that carry out extensive fatty acid synthesis(liver, adipose, lactating mammary gland) or very ac-tive synthesis of cholesterol and steroid hormones(liver, adrenal gland, gonads) require the NADPH pro-vided by the pathway Erythrocytes and the cells ofthe lens and cornea are directly exposed to oxygen andthus to the damaging free radicals generated by oxygen

14.5 Pentose Phosphate Pathway of Glucose Oxidation 549

Pyruvate

AlanineCysteineGlycineSerineThreonineTryptophan*

-Ketoglutarate

ArginineGlutamateGlutamineHistidineProline

Glucogenic Amino Acids, Grouped

by Site of Entry

Note: All these amino acids are precursors of blood glucose or liver glycogen, because they

can be converted to pyruvate or citric acid cycle intermediates Of the 20 common amino

acids, only leucine and lysine are unable to furnish carbon for net glucose synthesis.

*These amino acids are also ketogenic (see Fig 18–21).

TABLE 14–4

Succinyl-CoA

Isoleucine*

MethionineThreonineValine

By maintaining a reducing atmosphere (a high ratio of

NADPH to NADP and a high ratio of reduced to

oxi-dized glutathione), they can prevent or undo oxidative

damage to proteins, lipids, and other sensitive molecules

In erythrocytes, the NADPH produced by the pentose

phosphate pathway is so important in preventing

oxida-tive damage that a genetic defect in glucose 6-phosphate

dehydrogenase, the first enzyme of the pathway, can

have serious medical consequences (Box 14–3) ■

The Oxidative Phase Produces Pentose Phosphates

and NADPH

The first reaction of the pentose phosphate pathway

(Fig 14–21) is the oxidation of glucose 6-phosphate

by glucose 6-phosphate dehydrogenase (G6PD) to

form 6-phosphoglucono--lactone, an intramolecular

ester NADPis the electron acceptor, and the overall

equilibrium lies far in the direction of NADPH

forma-tion The lactone is hydrolyzed to the free acid

6-phos-phogluconate by a specific lactonase, then

6-phospho-gluconate undergoes oxidation and decarboxylation by

6-phosphogluconate dehydrogenase to form the

ke-topentose ribulose 5-phosphate This reaction generates

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

550

Nonoxidative

phase

Oxidative phase

NADP

NADPH

2 GSH

GSSG Fatty acids, sterols, etc.

Precursors

transketolase,

transaldolase

glutathione reductase

reductive biosynthesis

NADPNADPH

HOCH O

gluconate

6-Phospho-Glucose 6-phosphate

D -Ribose 5-phosphate

phosphopentose isomerase

glucose 6-phosphate dehydrogenase

6-phosphogluconate dehydrogenase

A

HC A

O

3

A HCOH

C HCOH HOCH

P A

HCOH

A HCOH

O A

CO 2

3

A

D -Ribulose 5-phosphate

Mg 2 

Mg 2 

glucono- -lactone

6-Phospho-NADP

NADPH

A HC

O

A

HCOH A

A HCOH A

HOCH A

HCOH A A HCOH

A

A HCOH CHO

A HCOH A

A HCOH NADPH

FIGURE 14–20 General scheme of the pentose phosphate pathway.

NADPH formed in the oxidative phase is used to reduce glutathione,

GSSG (see Box 14–3) and to support reductive biosynthesis The other

product of the oxidative phase is ribose 5-phosphate, which serves as

precursor for nucleotides, coenzymes, and nucleic acids In cells that

are not using ribose 5-phosphate for biosynthesis, the nonoxidative

phase recycles six molecules of the pentose into five molecules of the

hexose glucose 6-phosphate, allowing continued production of

NADPH and converting glucose 6-phosphate (in six cycles) to CO2.

FIGURE 14–21 Oxidative reactions of the pentose phosphate way The end products are ribose 5-phosphate, CO2 , and NADPH.

path-14.5 Pentose Phosphate Pathway of Glucose Oxidation 551

Why Pythagoras Wouldn’t Eat Falafel: Glucose 6-Phosphate Dehydrogenase Deficiency

Fava beans, an ingredient of falafel, have been an portant food source in the Mediterranean and MiddleEast since antiquity The Greek philosopher and math-ematician Pythagoras prohibited his followers fromdining on fava beans, perhaps because they makemany people sick with a condition called favism, whichcan be fatal In favism, erythrocytes begin to lyse 24

im-to 48 hours after ingestion of the beans, releasing freehemoglobin into the blood Jaundice and sometimeskidney failure can result Similar symptoms can occurwith ingestion of the antimalarial drug primaquine or

of sulfa antibiotics or following exposure to certainherbicides These symptoms have a genetic basis: glu-cose 6-phosphate dehydrogenase (G6PD) deficiency,which affects about 400 million people Most G6PD-deficient individuals are asymptomatic; only the com-bination of G6PD deficiency and certain environmen-tal factors produces the clinical manifestations

G6PD catalyzes the first step in the pentose phate pathway (see Fig 14–21), which producesNADPH This reductant, essential in many biosyn-thetic pathways, also protects cells from oxidativedamage by hydrogen peroxide (H2O2) and superoxidefree radicals, highly reactive oxidants generated asmetabolic byproducts and through the actions of drugssuch as primaquine and natural products such as di-vicine—the toxic ingredient of fava beans Duringnormal detoxification, H2O2is converted to H2O by re-duced glutathione and glutathione peroxidase, and theoxidized glutathione is converted back to the reducedform by glutathione reductase and NADPH (Fig 1)

phos-H2O2is also broken down to H2O and O2by catalase,which also requires NADPH In G6PD-deficientindividuals, the NADPH production is diminished anddetoxification of H2O2 is inhibited Cellular damageresults: lipid peroxidation leading to breakdown oferythrocyte membranes and oxidation of proteinsand DNA

The geographic distribution of G6PD deficiency isinstructive Frequencies as high as 25% occur in trop-ical Africa, parts of the Middle East, and SoutheastAsia, areas where malaria is most prevalent In addi-tion to such epidemiological observations, in vitro

studies show that growth of one malaria parasite, modium falciparum, is inhibited in G6PD-deficient

Plas-erythrocytes The parasite is very sensitive to tive damage and is killed by a level of oxidative stressthat is tolerable to a G6PD-deficient human host Be-cause the advantage of resistance to malaria balancesthe disadvantage of lowered resistance to oxidativedamage, natural selection sustains the G6PD-deficientgenotype in human populations where malaria isprevalent Only under overwhelming oxidative stress,caused by drugs, herbicides, or divicine, does G6PDdeficiency cause serious medical problems

oxida-An antimalarial drug such as primaquine is lieved to act by causing oxidative stress to the para-site It is ironic that antimalarial drugs can cause ill-ness through the same biochemical mechanism thatprovides resistance to malaria Divicine also acts as anantimalarial drug, and ingestion of fava beans may pro-tect against malaria By refusing to eat falafel, manyPythagoreans with normal G6PD activity may have un-wittingly increased their risk of malaria!

be-FIGURE 1 Role of NADPH and glutathione in protecting cells against highly reactive oxygen derivatives Reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hy- droxyl free radicals Regeneration of GSH from its oxidized form (GSSG) requires the NADPH produced in the glucose 6-phosphate dehydrogenase reaction.

Mitochondrial respiration, ionizing radiation, sulfa drugs, herbicides, antimalarials, divicine

Oxidative damage to lipids, proteins, DNA

Hydrogen peroxide

Hydroxyl free radical

NADP NADPHH

Glucose 6-phosphate

glucono-d-lactone

6-Phospho-glucose 6-phosphate dehydrogenase (G6PD)

glutathione reductase glutathione peroxidase

e

a second molecule of NADPH Phosphopentose

merase converts ribulose 5-phosphate to its aldose

iso-mer, ribose 5-phosphate In some tissues, the pentose

phosphate pathway ends at this point, and its overall

equation is

Glucose 6-phosphate  2NADP   H 2 O88n

ribose 5-phosphate  CO 2  2NADPH  2H The net result is the production of NADPH, a reductant

for biosynthetic reactions, and ribose 5-phosphate, a

precursor for nucleotide synthesis

The Nonoxidative Phase Recycles Pentose

Phosphates to Glucose 6-Phosphate

In tissues that require primarily NADPH, the pentose

phosphates produced in the oxidative phase of the

path-way are recycled into glucose 6-phosphate In this

non-oxidative phase, ribulose 5-phosphate is first epimerized

to xylulose 5-phosphate:

Then, in a series of rearrangements of the carbon

skele-tons (Fig 14–22), six five-carbon sugar phosphates are

CH 2 OH O C OH H

OH H

C C

CH2OPO3

CH 2 OH O C

OH H

C C

CH2OPO3

ribose 5-phosphate epimerase

Ribulose 5-phosphate

Xylulose 5-phosphate

converted to five six-carbon sugar phosphates, pleting the cycle and allowing continued oxidation ofglucose 6-phosphate with production of NADPH Con-tinued recycling leads ultimately to the conversion ofglucose 6-phosphate to six CO2 Two enzymes unique tothe pentose phosphate pathway act in these intercon-versions of sugars: transketolase and transaldolase

com-Transketolase catalyzes the transfer of a two-carbon

fragment from a ketose donor to an aldose acceptor(Fig 14–23a) In its first appearance in the pentosephosphate pathway, transketolase transfers C-1 and C-2 of xylulose 5-phosphate to ribose 5-phosphate,forming the seven-carbon product sedoheptulose 7-phosphate (Fig 14–23b) The remaining three-carbonfragment from xylulose is glyceraldehyde 3-phosphate

Next, transaldolase catalyzes a reaction similar to

the aldolase reaction of glycolysis: a three-carbon ment is removed from sedoheptulose 7-phosphate andcondensed with glyceraldehyde 3-phosphate, formingfructose 6-phosphate and the tetrose erythrose 4-phos-phate (Fig 14–24) Now transketolase acts again, form-ing fructose 6-phosphate and glyceraldehyde 3-phosphatefrom erythrose 4-phosphate and xylulose 5-phosphate(Fig 14–25) Two molecules of glyceraldehyde 3-phos-phate formed by two iterations of these reactions can beconverted to a molecule of fructose 1,6-bisphosphate as

frag-in gluconeogenesis (Fig 14–16), and ffrag-inally FBPase-1 andphosphohexose isomerase convert fructose 1,6-bisphos-phate to glucose 6-phosphate The cycle is complete: sixpentose phosphates have been converted to five hexosephosphates (Fig 14–22b)

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

552

Sedoheptulose 7-phosphate

fructose bisphosphatase

1,6-Glyceraldehyde 3-phosphate

Fructose 6-phosphate

Erythrose 4-phosphate

3-phosphate

Fructose 6-phosphate

Glucose 6-phosphate

aldolase triose phosphate isomerase

3C

6C

3C 5C

4C 3C

7C

3C

5C

5C 5C

FIGURE 14–22 Nonoxidative reactions of the pentose phosphate

pathway (a) These reactions convert pentose phosphates to hexose

phosphates, allowing the oxidative reactions (see Fig 14–21) to

con-tinue The enzymes transketolase and transaldolase are specific to this

pathway; the other enzymes also serve in the glycolytic or

gluco-neogenic pathways (b) A schematic diagram showing the pathway

from six pentoses (5C) to five hexoses (6C) Note that this involves two

sets of the interconversions shown in (a) Every reaction shown here

is reversible; unidirectional arrows are used only to make clear the direction of the reactions during continuous oxidation of glucose 6- phosphate In the light-independent reactions of photosynthesis, the direction of these reactions is reversed (see Fig 20–10).

14.5 Pentose Phosphate Pathway of Glucose Oxidation 553

C O

CHOH



Ketose donor

Aldose acceptor

TPP transketolase

(a)

CH2OH

R 2

C O

Ribose 5-phosphate

Glyceraldehyde 3-phosphate

Sedoheptulose 7-phosphate

TPP transketolase

(b)

C O

CH2OH

O C C

trans-on enzyme-bound TPP, from a ketose donor to an aldose acceptor.

(b) Conversion of two pentose

phosphates to a triose phosphate and

a seven-carbon sugar phosphate, sedoheptulose 7-phosphate.



Glyceraldehyde 3-phosphate

Erythrose 4-phosphate

Fructose 6-phosphate

transaldolase

O C

Erythrose 4-phosphate

Fructose 6-phosphate

transketolase

 C

CH 2 OPO 3

O C H

py-banion in this reaction (Fig 14–26a), just as it does in

the pyruvate decarboxylase reaction (Fig 14–13)

Transaldolase uses a Lys side chain to form a Schiff base

with the carbonyl group of its substrate, a ketose,

thereby stabilizing a carbanion (Fig 14–26b) that is tral to the reaction mechanism

cen-The process described in Figure 14–21 is known as

the oxidative pentose phosphate pathway The first

two steps are oxidations with large, negative standardfree-energy changes and are essentially irreversible in

the cell The reactions of the nonoxidative part of the

pentose phosphate pathway (Fig 14–22) are readily

re-versible and thus also provide a means of converting

hexose phosphates to pentose phosphates As we shall

see in Chapter 20, a process that converts hexose

phos-phates to pentose phosphos-phates is crucial to the

photo-synthetic assimilation of CO2by plants That pathway,

the reductive pentose phosphate pathway, is

es-sentially the reversal of the reactions shown in Figure

14–22 and employs many of the same enzymes

All the enzymes in the pentose phosphate pathway

are located in the cytosol, like those of glycolysis and

most of those of gluconeogenesis In fact, these three

pathways are connected through several shared

inter-mediates and enzymes The glyceraldehyde

3-phos-phate formed by the action of transketolase is readily

converted to dihydroxyacetone phosphate by the

gly-colytic enzyme triose phosphate isomerase, and these

two trioses can be joined by the aldolase as in

gluco-neogenesis, forming fructose 1,6-bisphosphate

Alterna-tively, the triose phosphates can be oxidized to

pyru-vate by the glycolytic reactions The fate of the trioses

is determined by the cell’s relative needs for pentose

phosphates, NADPH, and ATP

Wernicke-Korsakoff Syndrome Is Exacerbated by a

Defect in Transketolase

In humans with Wernicke-Korsakoff syndrome, amutation in the gene for transketolase results in

an enzyme having an affinity for its coenzyme TPP that

is one-tenth that of the normal enzyme Although erate deficiencies in the vitamin thiamine have little ef-fect on individuals with an unmutated transketolasegene, in those with the altered gene, thiamine deficiencydrops the level of TPP below that needed to saturatethe enzyme The lowering of transketolase activity slowsthe whole pentose phosphate pathway, and the result isthe Wernicke-Korsakoff syndrome: severe memory loss,mental confusion, and partial paralysis The syndrome

mod-is more common among alcoholics than in the generalpopulation; chronic alcohol consumption interferes withthe intestinal absorption of some vitamins, includingthiamine ■

Glucose 6-Phosphate Is Partitioned between Glycolysis and the Pentose Phosphate Pathway

Whether glucose 6-phosphate enters glycolysis or thepentose phosphate pathway depends on the currentneeds of the cell and on the concentration of NADP

in the cytosol Without this electron acceptor, the firstreaction of the pentose phosphate pathway (catalyzed

by G6PD) cannot proceed When a cell is rapidly verting NADPH to NADPin biosynthetic reductions,the level of NADP rises, allosterically stimulatingG6PD and thereby increasing the flux of glucose 6-phosphate through the pentose phosphate pathway(Fig 14–27) When the demand for NADPH slows, thelevel of NADPdrops, the pentose phosphate pathwayslows, and glucose 6-phosphate is instead used to fuelglycolysis

con-Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

554

Glucose

Glucose 6-phosphate

pentose phosphate pathway

glycolysis

gluconolactone

6-Phospho-Pentose phosphates

C

N H

resonance stabilization

Protonated Schiff base

FIGURE 14–26 Carbanion intermediates stabilized by covalent

in-teractions with transketolase and transaldolase (a) The ring of TPP

stabilizes the two-carbon carbanion carried by transketolase; see Fig.

14–13 for the chemistry of TPP action (b) In the transaldolase

reac-tion, the protonated Schiff base formed between the -amino group

of a Lys side chain and the substrate stabilizes a three-carbon

carbanion.

FIGURE 14–27 Role of NADPH in regulating the partitioning of cose 6-phosphate between glycolysis and the pentose phosphate pathway When NADPH is forming faster than it is being used for

glu-biosynthesis and glutathione reduction (see Fig 14–20), [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway.

As a result, more glucose 6-phosphate is available for glycolysis.

SUMMARY 14.5 Pentose Phosphate Pathway of

Glucose Oxidation

■ The oxidative pentose phosphate pathway

(phosphogluconate pathway, or hexosemonophosphate pathway) brings aboutoxidation and decarboxylation at C-1 of glucose6-phosphate, reducing NADPto NADPH andproducing pentose phosphates

■ NADPH provides reducing power forbiosynthetic reactions, and ribose 5-phosphate

is a precursor for nucleotide and nucleic acidsynthesis Rapidly growing tissues and tissuescarrying out active biosynthesis of fatty acids,cholesterol, or steroid hormones send moreglucose 6-phosphate through the pentosephosphate pathway than do tissues with lessdemand for pentose phosphates and reducingpower

■ The first phase of the pentose phosphatepathway consists of two oxidations that convertglucose 6-phosphate to ribulose 5-phosphate

and reduce NADPto NADPH The secondphase comprises nonoxidative steps thatconvert pentose phosphates to glucose 6-phosphate, which begins the cycle again

■ In the second phase, transaldolase (with TPP

as cofactor) and transketolase catalyze theinterconversion of three-, four-, five-, six-, andseven-carbon sugars, with the reversibleconversion of six pentose phosphates to fivehexose phosphates In the carbon-assimilatingreactions of photosynthesis, the same enzymescatalyze the reverse process, called the

reductive pentose phosphate pathway:

conversion of five hexose phosphates to sixpentose phosphates

■ A genetic defect in transketolase that lowers itsaffinity for TPP exacerbates the Wernicke-Korsakoff syndrome

■ Entry of glucose 6-phosphate either intoglycolysis or into the pentose phosphatepathway is largely determined by the relativeconcentrations of NADP and NADPH

Chapter 14 Further Reading 555

Terms in bold are defined in the glossary.

Key Terms

glycolysis 522

fermentation 522 lactic acid fermentation

hypoxia 523

ethanol (alcohol) fermentation 523

isozymes 526

acyl phosphate 530

substrate-level phorylation 531

respiration-linked phorylation 531 phosphoenolpyruvate (PEP) 532

phos-mutases 534

isomerases 534 lactose intolerance galactosemia 537

thiamine phate (TPP) 540

pyrophos-gluconeogenesis 543

biotin 544

pentose phosphate pathway 549

phosphogluconate pathway 549

hexose monophosphate pathway 549

Further Reading

General

Fruton, J.S (1999) Proteins, Genes, and Enzymes: The

Inter-play of Chemistry and Biology, Yale University Press, New Haven.

This text includes a detailed historical account of research on glycolysis.

Glycolysis

Boiteux, A & Hess, B (1981) Design of glycolysis Philos.

Trans R Soc Lond Ser B Biol Sci.293, 5–22

Intermediate-level review of the pathway and the classic view

of its control

Dandekar, T., Schuster, S., Snel, B., Huynen, M., & Bork, P.

(1999) Pathway alignment: application to the comparative analysis

of glycolytic enzymes Biochem J 343, 115–124.

Intermediate-level review of the bioinformatic view of the lution of glycolysis.

evo-Dang, C.V & Semenza, G.L (1999) Oncogenic alterations of

me-tabolism Trends Biochem Sci 24, 68–72.

Brief review of the molecular basis for increased glycolysis in tumors.

Erlandsen, H., Abola, E.E., & Stevens, R.C (2000) Combining

structural genomics and enzymology: completing the picture in

metabolic pathways and enzyme active sites Curr Opin Struct.

Biol.10, 719–730.

Intermediate-level review of the structures of the glycolytic enzymes.

Hardie, D.G (2000) Metabolic control: a new solution to an old

problem Curr Biol 10, R757–R759.

Harris, A.L (2002) Hypoxia—a key regulatory factor in tumour

growth Nat Rev Cancer 2, 38–47.

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

556

Heinrich, R., Melendez-Hevia, E., Montero, F., Nuno, J.C.,

Stephani, A., & Waddell, T.D (1999) The structural design of

glycolysis: an evolutionary approach Biochem Soc Trans 27,

294–298.

Knowles, J & Albery, W.J (1977) Perfection in enzyme

cataly-sis: the energetics of triose phosphate isomerase Acc Chem Res.

10, 105–111.

Phillips, D., Blake, C.C.F., & Watson, H.C (eds) (1981) The

Enzymes of Glycolysis: Structure, Activity and Evolution Philos.

Trans R Soc Lond Ser B Biol Sci.293, 1–214.

A collection of excellent reviews on the enzymes of glycolysis,

written at a level challenging but comprehensible to a

begin-ning student of biochemistry.

Plaxton, W.C (1996) The organization and regulation of plant

glycolysis Annu Rev Plant Physiol Plant Mol Biol 47,

185–214.

Very helpful review of the subcellular localization of glycolytic

enzymes and the regulation of glycolysis in plants.

Rose, I (1981) Chemistry of proton abstraction by glycolytic

en-zymes (aldolase, isomerases, and pyruvate kinase) Philos Trans.

R Soc Lond Ser B Biol Sci 293, 131–144.

Intermediate-level review of the mechanisms of these enzymes.

Shirmer, T & Evans, P.R (1990) Structural basis for the

al-losteric behavior of phosphofructokinase Nature 343, 140–145.

Smith, T.A (2000) Mammalian hexokinases and their abnormal

expression in cancer Br J Biomed Sci 57, 170–178.

A review of the four hexokinase isozymes of mammals: their

properties and tissue distributions and their expression during

the development of tumors.

Feeder Pathways for Glycolysis

Elsas, L.J & Lai, K (1998) The molecular biology of

galac-tosemia Genet Med 1, 40–48.

Novelli, G & Reichardt, J.K (2000) Molecular basis of

disor-ders of human galactose metabolism: past, present, and future.

Mol Genet Metab.71, 62–65.

Petry, K.G & Reichardt, J.K (1998) The fundamental

impor-tance of human galactose metabolism: lessons from genetics and

biochemistry Trends Genet 14, 98–102.

Van Beers, E.H., Buller, H.A., Grand, R.J., Einerhand,

A.W.C., & Dekker, J (1995) Intestinal brush border

glycohydro-lases: structure, function, and development Crit Rev Biochem.

Mol Biol.30, 197–262.

Fermentations

Behal, R.H., Buxton, D.B., Robertson, J.G., & Olson, M.S.

(1993) Regulation of the pyruvate dehydrogenase multienzyme

complex Annu Rev Nutr 13, 497–520.

Patel, M.S., Naik, S., Wexler, I.D., & Kerr, D.S (1995) Gene

regulation and genetic defects in the pyruvate dehydrogenase

com-plex J Nutr 125, 1753S–1757S.

Patel, M.S & Roche, T.E (1990) Molecular biology and

bio-chemistry of pyruvate dehydrogenase complexes FASEB J 4,

3224–3233.

Robinson, B.H., MacKay, N., Chun, K., & Ling, M (1996)

Dis-orders of pyruvate carboxylase and the pyruvate dehydrogenase

complex J Inherit Metab Dis 19, 452–462

Gluconeogenesis

Gerich, J.E., Meyer, C., Woerle, H.J., & Stumvoll, M (2001)

Renal gluconeogenesis: its importance in human glucose

homeosta-sis Diabetes Care 24, 382–391.

Intermediate-level review of the contribution of kidney tissue to gluconeogenesis

Gleeson, T (1996) Post-exercise lactate metabolism: a

compara-tive review of sites, pathways, and regulation Annu Rev Physiol.

58, 565–581.

Hers, H.G & Hue, L (1983) Gluconeogenesis and related

as-pects of glycolysis Annu Rev Biochem 52, 617–653.

Matte, A., Tari, L.W., Goldie, H., & Delbaere, L.T.J (1997)

Structure and mechanism of phosphoenolpyruvate carboxykinase.

J Biol Chem.272, 8105–8108.

Oxidative Pentose Phosphate Pathway

Chayen, J., Howat, D.W., & Bitensky, L (1986) Cellular

bio-chemistry of glucose 6-phosphate and 6-phosphogluconate

dehy-drogenase activities Cell Biochem Funct 4, 249–253.

Horecker, B.L (1976) Unraveling the pentose phosphate

path-way In Reflections on Biochemistry (Kornberg, A., Cornudella,

L., Horecker, B.L., & Oro, J., eds), pp 65–72, Pergamon Press, Inc., Oxford.

Kletzien, R.F., Harris, P.K., & Foellmi, L.A (1994) Glucose

6-phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant

stress FASEB J 8, 174–181.

An intermediate-level review.

Luzzato, L., Mehta, A., & Vulliamy, T (2001) Glucose

6-phos-phate dehydrogenase deficiency In The Metabolic and Molecular

Bases of Inherited Disease, 8th edn (Scriver, C.R., Sly, W.S.,

Childs, B., Beaudet, A.L., Valle, D., Kinzler, K.W., & Vogelstein, B., eds), pp 4517–4553, McGraw-Hill Inc., New York.

The four-volume treatise in which this article appears is filled with fascinating information about the clinical and biochemical features of hundreds of inherited diseases of metabolism.

Martini, G & Ursini, M.V (1996) A new lease on life for an old

enzyme BioEssays 18, 631–637.

An intermediate-level review of glucose 6-phosphate genase, the effects of mutations in this enzyme in humans, and the effects of knock-out mutations in mice.

dehydro-Notaro, R., Afolayan, A., & Luzzatto, L (2000) Human

muta-tions in glucose 6-phosphate dehydrogenase reflect evolutionary

history FASEB J 14, 485–494.

Wood, T (1985) The Pentose Phosphate Pathway, Academic

Press, Inc., Orlando, FL.

Wood, T (1986) Physiological functions of the pentose phosphate

pathway Cell Biochem Funct 4, 241–247.

Chapter 14 Problems 557

1 Equation for the Preparatory Phase of Glycolysis

Write balanced biochemical equations for all the reactions in

the catabolism of glucose to two molecules of glyceraldehyde

3-phosphate (the preparatory phase of glycolysis), including

the standard free-energy change for each reaction Then write

the overall or net equation for the preparatory phase of

gly-colysis, with the net standard free-energy change.

2 The Payoff Phase of Glycolysis in Skeletal Muscle

In working skeletal muscle under anaerobic conditions,

glyc-eraldehyde 3-phosphate is converted to pyruvate (the payoff

phase of glycolysis), and the pyruvate is reduced to lactate.

Write balanced biochemical equations for all the reactions in

this process, with the standard free-energy change for each

reaction Then write the overall or net equation for the

pay-off phase of glycolysis (with lactate as the end product),

in-cluding the net standard free-energy change.

3 Pathway of Atoms in Fermentation A “pulse-chase”

experiment using 14 C-labeled carbon sources is carried out

on a yeast extract maintained under strictly anaerobic

con-ditions to produce ethanol The experiment consists of

incu-bating a small amount of 14C-labeled substrate (the pulse)

with the yeast extract just long enough for each

intermedi-ate in the fermentation pathway to become labeled The

la-bel is then “chased” through the pathway by the addition of

excess unlabeled glucose The chase effectively prevents any

further entry of labeled glucose into the pathway.

(a) If [1-14C]glucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of 14 C in the prod-

uct ethanol? Explain.

(b) Where would 14 C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO 2

during fermentation to ethanol? Explain.

4 Fermentation to Produce Soy Sauce Soy sauce is prepared by fermenting a salted mixture of soybeans and

wheat with several microorganisms, including yeast, over a

period of 8 to 12 months The resulting sauce (after solids

are removed) is rich in lactate and ethanol How are these

two compounds produced? To prevent the soy sauce from

having a strong vinegar taste (vinegar is dilute acetic acid),

oxygen must be kept out of the fermentation tank Why?

5 Equivalence of Triose Phosphates 14C-Labeled glyceraldehyde 3-phosphate was added to a yeast extract.

After a short time, fructose 1,6-bisphosphate labeled with

14 C at C-3 and C-4 was isolated What was the location of the

mu-the presence of a new enzyme catalyzing mu-the reaction:

Would shortening the glycolytic pathway in this way benefit the cell? Explain.

7 Role of Lactate Dehydrogenase During strenuous tivity, the demand for ATP in muscle tissue is vastly increased.

ac-In rabbit leg muscle or turkey flight muscle, the ATP is duced almost exclusively by lactic acid fermentation ATP is formed in the payoff phase of glycolysis by two reactions, pro- moted by phosphoglycerate kinase and pyruvate kinase Sup- pose skeletal muscle were devoid of lactate dehydrogenase Could it carry out strenuous physical activity; that is, could

pro-it generate ATP at a high rate by glycolysis? Explain.

8 Efficiency of ATP Production in Muscle The formation of glucose to lactate in myocytes releases only about 7% of the free energy released when glucose is completely ox- idized to CO 2 and H 2 O Does this mean that anaerobic glycol- ysis in muscle is a wasteful use of glucose? Explain.

trans-9 Free-Energy Change for Triose Phosphate Oxidation

The oxidation of glyceraldehyde 3-phosphate to phoglycerate, catalyzed by glyceraldehyde 3-phosphate dehy- drogenase, proceeds with an unfavorable equilibrium constant

1,3-bisphos-(K eq 0.08; G  6.3 kJ/mol), yet the flow through this

point in the glycolytic pathway proceeds smoothly How does the cell overcome the unfavorable equilibrium?

10 Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate (P i ), and many en- zymes that require phosphate will also use arsenate Organic compounds of arsenate are less stable than analogous phos-

phate compounds, however For example, acyl arsenates

de-compose rapidly by hydrolysis:

On the other hand, acyl phosphates, such as

1,3-bisphos-phoglycerate, are more stable and undergo further catalyzed transformation in cells.

enzyme-(a) Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate.

(b) What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms Explain why.

11 Requirement for Phosphate in Ethanol tion In 1906 Harden and Young, in a series of classic stud- ies on the fermentation of glucose to ethanol and CO 2 by extracts of brewer’s yeast, made the following observations (1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used (2) During fermentation un- der these conditions, ethanol, CO2, and a hexose bisphosphate

O B

O

O

As O C

O

O B

Glyceraldehyde 3-phosphate  H 2

3-phosphoglycerate NAD NADH  H 

Problems

accumulated (3) When arsenate was substituted for

phos-phate, no hexose bisphosphate accumulated, but the

fer-mentation proceeded until all the glucose was converted to

ethanol and CO2.

(a) Why did fermentation cease when the supply of

phosphate was exhausted?

(b) Why did ethanol and CO2accumulate? Was the

con-version of pyruvate to ethanol and CO2essential? Why?

Iden-tify the hexose bisphosphate that accumulated Why did it

accumulate?

(c) Why did the substitution of arsenate for phosphate

prevent the accumulation of the hexose bisphosphate yet

al-low fermentation to ethanol and CO2 to go to completion?

(See Problem 10.)

12 Role of the Vitamin Niacin Adults engaged in

stren-uous physical activity require an intake of about 160 g of

car-bohydrate daily but only about 20 mg of niacin for optimal

nutrition Given the role of niacin in glycolysis, how do you

explain the observation?

13 Metabolism of Glycerol Glycerol obtained from the

breakdown of fat is metabolized by conversion to

dihydroxy-acetone phosphate, a glycolytic intermediate, in two

enzyme-catalyzed reactions Propose a reaction sequence for glycerol

metabolism On which known enzyme-catalyzed reactions is

your proposal based? Write the net equation for the

conver-sion of glycerol to pyruvate according to your scheme.

14 Severity of Clinical Symptoms Due to Enzyme Deficiency The clinical symptoms of two forms of galactosemia—deficiency of galactokinase or

of UDP-glucose:galactose 1-phosphate uridylyltransferase—

show radically different severity Although both types

pro-duce gastric discomfort after milk ingestion, deficiency of the

transferase also leads to liver, kidney, spleen, and brain

dys-function and eventual death What products accumulate in

the blood and tissues with each type of enzyme deficiency?

Estimate the relative toxicities of these products from the

above information.

15 Muscle Wasting in Starvation One consequence of

starvation is a reduction in muscle mass What happens to

the muscle proteins?

16 Pathway of Atoms in Gluconeogenesis A liver

ex-tract capable of carrying out all the normal metabolic

reac-tions of the liver is briefly incubated in separate experiments

with the following 14 C-labeled precursors:

Trace the pathway of each precursor through sis Indicate the location of 14 C in all intermediates and in the product, glucose.

gluconeogene-17 Pathway of CO2 in Gluconeogenesis In the first pass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate, pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently decar- boxylated by PEP carboxykinase to yield phosphoenolpyru- vate The observation that the addition of CO2is directly fol- lowed by the loss of CO2 suggests that 14 C of 14 CO2would not be incorporated into PEP, glucose, or any intermediates

by-in gluconeogenesis However, when a rat liver preparation synthesizes glucose in the presence of 14CO2, 14C slowly ap- pears in PEP and eventually at C-3 and C-4 of glucose How does the 14C label get into PEP and glucose? (Hint: During gluconeogenesis in the presence of 14 CO2, several of the four- carbon citric acid cycle intermediates also become labeled.)

18 Energy Cost of a Cycle of Glycolysis and neogenesis What is the cost (in ATP equivalents) of trans- forming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis?

Gluco-19 Glucogenic Substrates A common procedure for termining the effectiveness of compounds as precursors of glucose in mammals is to starve the animal until the liver glycogen stores are depleted and then administer the com-

de-pound in question A substrate that leads to a net increase in

liver glycogen is termed glucogenic, because it must first be converted to glucose 6-phosphate Show by means of known enzymatic reactions which of the following substances are glucogenic:

20 Ethanol Affects Blood Glucose Levels The consumption of alcohol (ethanol), especially after pe- riods of strenuous activity or after not eating for several hours, results in a deficiency of glucose in the blood, a con- dition known as hypoglycemia The first step in the metabo- lism of ethanol by the liver is oxidation to acetaldehyde, cat- alyzed by liver alcohol dehydrogenase:

CH 3 CH 2 OH
NAD
88nCH 3 CHO
NADH
H
Explain how this reaction inhibits the transformation of lac- tate to pyruvate Why does this lead to hypoglycemia?

(a) Succinate,  OOC CH2 CH2

(b) Glycerol,

CH2

OH C OH

H

CH2OH

(c) Acetyl-CoA,

CH3 C S-CoA

(d) Pyruvate,

CH3 C O O

H

Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

558

Chapter 14 Problems 559

21 Blood Lactate Levels during Vigorous Exercise

The concentrations of lactate in blood plasma before, during,

and after a 400 m sprint are shown in the graph.

(a) What causes the rapid rise in lactate concentration?

(b) What causes the decline in lactate concentration ter completion of the sprint? Why does the decline occur more

af-slowly than the increase?

(c) Why is the concentration of lactate not zero during the resting state?

22 Relationship between Fructose 1,6-Bisphosphatase

and Blood Lactate Levels A congenital defect in the liver

enzyme fructose 1,6-bisphosphatase results in abnormally

high levels of lactate in the blood plasma Explain.

Time (min)

100

50

60 40

23 Effect of Phloridzin on Carbohydrate Metabolism

Phloridzin, a toxic glycoside from the bark of the pear tree, blocks the normal reabsorption of glucose from the kidney tubule, thus causing blood glucose to be almost completely excreted in the urine In an experiment, rats fed phloridzin and sodium succinate excreted about 0.5 mol of glucose (made by gluconeogenesis) for every 1 mol of sodium succi- nate ingested How is the succinate transformed to glucose? Explain the stoichiometry.

24 Excess O2 Uptake during Gluconeogenesis Lactate absorbed by the liver is converted to glucose, with the input

of 6 mol of ATP for every mole of glucose produced The tent of this process in a rat liver preparation can be moni- tored by administering [ 14 C]lactate and measuring the amount

ex-of [14C]glucose produced Because the stoichiometry of O2consumption and ATP production is known (about 5 ATP per

O2), we can predict the extra O2consumption above the mal rate when a given amount of lactate is administered How- ever, when the extra O2used in the synthesis of glucose from lactate is actually measured, it is always higher than predicted

nor-by known stoichiometric relationships Suggest a possible planation for this observation.

HOCH 2

CH 2

H O

Phloridzin

CH 2

c h a p t e r

Metabolic regulation, a central theme in

biochem-istry, is one of the most remarkable features of a

living cell Of the thousands of enzyme-catalyzed

reac-tions that can take place in a cell, there is probably not

one that escapes some form of regulation Although it

is convenient (and perhaps essential) in writing a book to divide metabolic processes into “pathways” thatplay discrete roles in the cell’s economy, no such sepa-ration exists inside the cell Rather, each of the path-ways we discuss in this book is inextricably intertwinedwith all the other cellular pathways in a multidimen-sional network of reactions (Fig 15–1) For example, inChapter 14 we discussed three possible fates for glu-cose 6-phosphate in a hepatocyte: passage into glycol-ysis for the production of ATP, passage into the pentosephosphate pathway for the production of NADPH andpentose phosphates, or hydrolysis to glucose and phos-phate to replenish blood glucose In fact, glucose 6-phos-phate has a number of other possible fates; it may, forexample, be used to synthesize other sugars, such asglucosamine, galactose, galactosamine, fucose, and neu-raminic acid, for use in protein glycosylation, or it may

text-be partially degraded to provide acetyl-CoA for fattyacid and sterol synthesis In the extreme case, the bac-

terium Escherichia coli can use glucose to produce the

carbon skeleton of every one of its molecules When acell “decides” to use glucose 6-phosphate for one pur-pose, that decision affects all the other pathways forwhich glucose 6-phosphate is a precursor or intermedi-ate; any change in the allocation of glucose 6-phosphate

to one pathway affects, directly or indirectly, themetabolite flow through all the others

Such changes in allocation are common in the life

of a cell Louis Pasteur was the first to describe the large(greater than tenfold) increase in glucose consumption

by a yeast culture when it was shifted from aerobic toanaerobic conditions This phenomenon, called the

PRINCIPLES OF METABOLIC

REGULATION: GLUCOSE AND

GLYCOGEN

15.1 The Metabolism of Glycogen in Animals 562

15.2 Regulation of Metabolic Pathways 571

15.3 Coordinated Regulation of Glycolysis and

Gluconeogenesis 575

15.4 Coordinated Regulation of Glycogen Synthesis and

Breakdown 583

15.5 Analysis of Metabolic Control 591

Formation of liver glycogen from lactic acid is thus seen

to establish an important connection between the

metabolism of the muscle and that of the liver Muscle

glycogen becomes available as blood sugar through the

intervention of the liver, and blood sugar in turn is

converted into muscle glycogen There exists therefore a

complete cycle of the glucose molecule in the body

Epinephrine was found to accelerate this cycle in the

direction of muscle glycogen to liver glycogen Insulin,

on the other hand, was found to accelerate the cycle in

the direction of blood glucose to muscle glycogen

—C F Cori and G T Cori, article in Journal of

Biological Chemistry, 1929

560

15